WO2022120936A1 - Modified nucleic acid and application thereof - Google Patents

Modified nucleic acid and application thereof Download PDF

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WO2022120936A1
WO2022120936A1 PCT/CN2020/138025 CN2020138025W WO2022120936A1 WO 2022120936 A1 WO2022120936 A1 WO 2022120936A1 CN 2020138025 W CN2020138025 W CN 2020138025W WO 2022120936 A1 WO2022120936 A1 WO 2022120936A1
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fluoro
nucleic acid
modified
deoxy
modified nucleic
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French (fr)
Chinese (zh)
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胡勇
张苗苗
洪丹
胡迅
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深圳市瑞吉生物科技有限公司
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Definitions

  • the invention belongs to the technical field of nucleic acid modification, and in particular relates to a modified nucleic acid and its application.
  • m6A modification is related to mRNA stability, splicing processing, translation, and microRNA processing.
  • m6A is also related to stem cell fate and biological rhythm, and can promote stem cells from a state of self-renewal to cell differentiation. The researchers found that methylation shortens the half-life of mRNA and reduces its abundance. It can be said that m6A modification affects almost every step of RNA metabolism.
  • RNA synthesis mainly include the following:
  • 2'-O-methylation (Nm) Regarding the 2'-O-methylation modification (Nm) of the 5' cap, it is generally believed that the 5' cap can promote the binding of mRNA and ribosome, and can effectively block RNA 5' end to protect mRNA from 5' exonuclease degradation and enhance mRNA stability. In addition, the 5' cap also participates in the splicing of mRNA precursors, participates in the polyadenylation of the 3' end of mRNA, and the transport of mRNA from the nucleus to the cytoplasm also requires the participation of the 5' cap. In 1998, Wei et al.
  • cap binding protein can interact with ploy(A) binding protein (PABP), shortening the distance between the 5' cap and the tail of ploy(A), and the formed ring structure can Strengthen the affinity of the cap structure and CBP, speed up the ribosome recycling, thereby improving the translation efficiency (Wei, C.-C., Balasta, M.L., Ren, J. & Goss, D.J. (1998). Wheat germ poly(A) binding protein enhances the binding affinity of eukaryotic initiation factor 4F and (iso)4F for cap analogies. Biochemistry 37, 1910-1916.). In 2010, Daffis et al.
  • the 2'-O-methylation modification of the 5' cap of viral RNA can allow it to escape the host's antiviral response, and the 2'-O-methylation modification of the 5' cap of cytoplasmic RNA may be the host to distinguish its own RNA. and an important marker of foreign RNA (Daffis, S., Szretter, K.J., Schriewer, J. & other authors (2010). 2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature 468, 452-456. ).
  • Mammalian cells are innately immune to viruses without 2'-O-methylation in their 5' caps, because IFIT1 (interferon-induced protein with tetratricopetide repeats 1, interferon-induced tetrapeptide repeats 1) can interact with such Viral mRNA binds so that it cannot be translated.
  • IFIT1 interferon-induced protein with tetratricopetide repeats 1, interferon-induced tetrapeptide repeats 1
  • Pseudouracil ( ⁇ ) Pseudouracil modification is the most abundant RNA modification, generally produced by the isomerization of uridine. Previous studies have shown that pseudouracil modification of mRNA has three main functions: changing codons, Enhances transcript stability and stress response. The process of mRNA pseudouracil modification is catalyzed by pseudouracil synthases (PUS), which changes the chemical structure of uracil nucleotides (U) to form pseudouracil nucleotides. Previous studies have found a large number of pseudouracils in tRNA, rRNA, and snRNA, and recent studies have confirmed that pseudouracils also exist in mRNA.
  • PUS pseudouracil synthases
  • RNA modifications that lack corresponding technical means to study in depth. Therefore, in terms of technology, more high-throughput technologies of the "NGS+" type (combination of traditional detection methods and high-throughput sequencing) are needed.
  • nanopore technology a novel single-molecule approach, has shown single-base resolution detection of m6A, and scientists believe that this single-molecule approach may become a novel paradigm to simultaneously detect different RNA modifications .
  • the object of the present invention is to provide a modified nucleic acid and its application, the modified nucleic acid is prepared by artificial synthesis, and the modified nucleic acid is obtained by synthesizing several chemically modified nucleotides, and the modified nucleic acid is obtained by synthesizing several chemically modified nucleotides.
  • the nucleic acid has high stability, low immunogenicity and long in vivo half-life; it has a wide range of applications.
  • the present invention provides a modified nucleic acid, including uridine, cytosine, adenosine, guanosine and chemically modified nucleosides; the chemically modified nucleosides include chemically modified uracil One or more of nucleosides, chemically modified cytosines, chemically modified adenosines and chemically modified guanosines.
  • the present invention provides a modified nucleic acid, including chemically modified nucleosides; the chemically modified nucleosides include chemically modified uridine nucleosides, chemically modified cytosine nucleosides, chemically modified adenosine and chemically modified nucleosides Modified guanosine.
  • the modified nucleic acid is ribonucleic acid.
  • the chemical modification in the chemically modified nucleosides includes isomerism and/or group substitution; the group substitution includes methyl substitution, methoxy substitution, halogen substitution and N4-acetyl substitution one or more of them.
  • the chemically modified uridine is selected from 2-fluoro-2-deoxyuridine, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 2-fluoro-2 - one or more of deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methoxyuridine.
  • the chemically modified cytidine is selected from N4-acetylcytidine, 2-fluoro-2-deoxycytidine, 5-methylcytidine, 2-fluoro-2-deoxy-5-methylcytidine One or more of cytidine and 2-fluoro-2-deoxy-N4-acetylcytidine.
  • the chemically modified adenosine is selected from one of N6-methyladenosine, 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N6-methyladenosine or several.
  • the chemically modified guanosine is selected from one of 2-fluoro-2-deoxyguanosine, N7-methyl-guanosine and 2-fluoro-2-deoxy-N7-methyl-guanosine species or several.
  • the 5'UTR, 3'UTR and 5'cap structure of the Kozak sequence are also included.
  • a poly-A tail is also included.
  • the modified nucleic acid is modified mRNA.
  • mRNA encoding the viral spike protein is included.
  • mRNAs encoding proteases and protein hormones in organisms are included.
  • the present invention provides the use of the modified nucleic acid in the preparation of a disease diagnostic agent and/or a therapeutic agent.
  • the present invention provides the application of the modified nucleic acid in the preparation of vaccine.
  • the present invention provides a pharmaceutical formulation comprising the modified nucleic acid and an excipient.
  • the excipient is selected from one of physiological saline, citrate buffer and citrate-physiological saline buffer.
  • the modified nucleic acid provided by the present invention is obtained by artificially synthesizing several modified nucleotides to obtain a modified nucleic acid with a specific sequence, and the modified nucleic acid has high stability, low immunogenicity and long in vivo half-life; the present invention provides The modified nucleic acid can be used as a diagnostic or therapeutic agent for the diagnosis and treatment of diseases. Compared with the existing nucleic acid in its natural state, it overcomes the problems of low stability, high immunogenicity, short half-life in vivo, and the need for repeated cycles in a short time. The disadvantages of drug administration, high cost, etc., reduce the application cost while enhancing the efficacy of nucleic acid drugs.
  • the binding of the modified erythropoietin (EPO) mRNA provided by the present invention to TRL3, TRL7, TRL8 and RIG-1 is significantly reduced compared to the unmodified EPO mRNA, and the levels of TNF ⁇ and IL-8 are also significantly reduced.
  • Modified mRNAs are significantly more efficient than single species of modified mRNAs.
  • the single-type modified and multi-type modified mRNAs provided by the present invention significantly reduce the binding of Toll-like receptors, thereby reducing the immune response, indicating that the modified mRNAs are less immunogenic than unmodified mRNAs, which are beneficial for in vivo applications.
  • modified nucleic acids provided by the present invention such as modified erythropoietin (EPO) mRNA and modified luciferase (Luc) mRNA, have higher expression levels in mice than the corresponding unmodified mRNAs , the expression time is longer, the expression is more stable, and can play a corresponding role, wherein the effect of multiple types of modified mRNA is better than that of a single type of modification.
  • EPO modified erythropoietin
  • Luc modified luciferase
  • the modified nucleic acid is mixed with excipients to obtain the pharmaceutical preparation.
  • the modified nucleic acid has better stability in terms of purity, concentration and pH value, and is convenient for storage. .
  • Figure 1 shows the binding level of a single chemically modified mRNA to the intracellular toll-like receptor TRL3 in cells
  • Figures 2 to 9 show the intracellular binding levels of various chemically modified mRNAs to the intracellular toll-like receptor TRL3;
  • Figure 10 shows the binding level of a single chemically modified mRNA to the intracellular toll-like receptor TRL7 in cells
  • Figures 11 to 18 show the intracellular binding levels of various chemically modified mRNAs to the intracellular toll-like receptor TRL7;
  • Figure 19 shows the binding level of a single chemically modified mRNA to the intracellular toll-like receptor TRL8 in cells
  • Figures 20-27 show the binding levels of various chemically modified mRNAs to the intracellular toll-like receptor TRL8 in cells
  • Figure 28 shows the binding level of a single chemically modified mRNA to the intracellular toll-like receptor RIG-1 in cells
  • Figures 29 to 36 are the intracellular binding levels of various chemically modified mRNAs to the intracellular toll-like receptor RIG-1;
  • Figure 37 shows the changes in the content of IL-8 in mouse serum after a single chemically modified mRNA was injected into mice;
  • Figures 38 to 44 show the changes in the content of IL-8 in the serum of mice after injection of various types of chemically modified mRNA into mice;
  • Figure 45 shows the changes in the content of TNF ⁇ in mouse serum after a single chemically modified mRNA was injected into mice;
  • Figures 46 to 53 show the changes in the content of TNF ⁇ in the serum of mice after injection of various chemically modified mRNAs into mice;
  • Figure 54 shows the expression of luciferase in mice after injection of a single chemically modified mRNA into mice
  • Figures 55 to 62 show the expression of luciferase in mice after various types of chemically modified mRNAs were injected into mice;
  • Figure 63 shows the expression of erythropoietin (EPO) in mouse serum after a single chemically modified mRNA was injected into mice;
  • Figures 64 to 71 show the content of erythropoietin (EPO) in the serum of mice after injection of various chemically modified mRNAs into mice;
  • Figure 72 shows the hematocrit of mice after injection of a single chemically modified mRNA into mice
  • Figures 73 to 80 show the hematocrit of mice after injection of various chemically modified mRNAs into mice
  • Figure 81 is the effect of different excipients on pH under mRNA drug storage conditions
  • Figure 82 is the effect of different excipients on the purity of mRNA drug storage conditions
  • Figure 83 is the effect of different excipients on the concentration of mRNA drug storage conditions
  • Figure 84 shows the relationship between the incorporation ratio of modified bases and the expression level of luciferase (Luc) mRNA
  • Figure 85 shows the effects of partial base modifications and all base modifications on the mRNA expression level of the spike protein (S) of the novel coronavirus 2019-nCov.
  • Figure 86 shows the results of the new coronavirus RBD mRNA expression protein concentration results for different chemical modification strategies
  • Figure 87 shows the antibody titer results of the new coronavirus RBD mRNA expression protein for different chemical modification strategies
  • Figure 88 shows the expression results of human transforming growth factor TGF ⁇ 3 mRNA with different chemical modification strategies
  • A1 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxyadenosine;
  • A2 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxycytidine;
  • A3 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxyguanosine;
  • A4 is the combined modification of 2-fluoro-2-deoxyuridine and 5-methylcytidine
  • A5 is the combined modification of 2-fluoro-2-deoxyuridine and N7-methyl-guanosine
  • A6 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-5-methylcytidine;
  • A7 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • A8 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-N4-acetylcytidine;
  • A9 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • B1 is the combined modification of pseudouridine and 2-fluoro-2-deoxyadenosine
  • B2 is the combined modification of pseudouridine and 2-fluoro-2-deoxycytidine
  • B3 is the combined modification of pseudouridine and 2-fluoro-2-deoxyguanosine
  • B4 is the combined modification of pseudouridine and 5-methylcytidine
  • B5 is the combined modification of pseudouridine and N7-methyl-guanosine
  • C1 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxyadenosine;
  • C2 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxycytidine;
  • C3 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxyguanosine;
  • C4 is the combined modification of N1-methyl-pseudouridine and 5-methylcytidine;
  • C5 is the combined modification of N1-methyl-pseudouridine and N7-methyl-guanosine;
  • C6 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methylcytidine;
  • C7 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • C8 is the combined modification of N1-methyl-pseudouridine 2-fluoro-2-deoxy-N4-acetylcytidine;
  • C9 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • D1 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxyadenosine
  • D2 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxycytidine
  • D3 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxyguanosine
  • D4 is the combined modification of 5-methoxyuridine and 5-methylcytidine
  • D5 is the combined modification of 5-methoxyuridine and N7-methyl-guanosine
  • D6 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-5-methylcytidine
  • D7 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • D8 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-N4-acetylcytidine;
  • D9 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-N6-methyladenosine
  • E1 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxyadenosine;
  • E2 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxyguanosine;
  • E3 is the combined modification of N4-acetylcytidine and N7-methyl-guanosine;
  • E4 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-pseudouridine;
  • E5 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • E6 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • E7 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-5-methoxyuridine;
  • E8 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • F1 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxycytidine
  • F2 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxyguanosine
  • F3 is the combined modification of N6-methyladenosine and 5-methylcytidine
  • F4 is the combined modification of N6-methyladenosine and N7-methyl-guanosine
  • F5 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-5-methylcytidine
  • F6 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-pseudouridine;
  • F7 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • F8 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • F9 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-5-methoxyuridine;
  • F10 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
  • G1 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-pseudouridine;
  • G2 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • G3 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • G4 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-5-methoxyuridine;
  • G5 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • G6 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-5-methylcytidine;
  • G7 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-pseudouridine;
  • G8 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • G9 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-5-methoxyuridine;
  • G10 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
  • G11 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • H1 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-5-methylcytidine;
  • H2 is a combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-pseudouridine;
  • H3 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • H4 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • H5 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-5-methoxyuridine;
  • H6 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
  • H7 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-pseudouridine
  • H8 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • H9 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • H10 is the combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-5-methoxyuridine
  • H11 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • H12 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-5-methylcytidine;
  • H13 is a combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-pseudouridine;
  • H14 is a combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • H15 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-5-methoxyuridine;
  • H16 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
  • H17 is a combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-N6-methyladenosine.
  • the present invention provides a modified nucleic acid, including uridine, cytosine, adenosine, guanosine and chemically modified nucleosides; the chemically modified nucleosides include chemically modified uracil One or more of nucleosides, chemically modified cytosines, chemically modified adenosines and chemically modified guanosines.
  • the specific sequence of the modified nucleic acid is not particularly limited in the present invention, and any sequence of nucleic acid is acceptable; the nucleic acid may encode known proteases and protein hormones in the organism; it may also encode non-existent or unknown in vivo encoding protease; the nucleic acid also includes mRNA encoding a viral spike protein; the nucleic acid preferably encodes a disease-associated protein.
  • the present invention does not specifically limit the type and quantity of chemically modified nucleosides in the modified nucleic acid; in a modified nucleic acid, there may be 1 to 4 kinds of modified nucleosides (including chemically modified uridine, chemically modified cytosine nucleosides, chemically modified adenosine nucleosides and chemically modified guanosine nucleosides); for a certain nucleoside, there can be different kinds of chemical modifications in a modified nucleic acid; for a certain nucleoside There can be different numbers of modification sites in a modified nucleic acid.
  • modified nucleosides including chemically modified uridine, chemically modified cytosine nucleosides, chemically modified adenosine nucleosides and chemically modified guanosine nucleosides
  • modified nucleosides including chemically modified uridine, chemically modified cytosine nucleosides, chemically modified adenosine nucleosides and
  • the chemical modification in the chemically modified nucleosides includes isomerization and/or group substitution; the group substitution includes methyl substitution, methoxy substitution, halogenation and N4-acetyl group one or more of the substitutions. In the present invention, the chemical modification also includes deoxygenation.
  • the chemically modified uridine is preferably selected from 2-fluoro-2-deoxyuridine, pseudouridine, N1-methyl-pseuduridine, 5-methyluridine oxyuridine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methoxyuridine one or more of them.
  • the chemically modified cytidine is selected from N4-acetylcytidine, 2-fluoro-2-deoxycytidine, 5-methylcytidine, 2-fluoro-cytidine One or more of 2-deoxy-5-methylcytidine and 2-fluoro-2-deoxy-N4-acetylcytidine.
  • the chemically modified adenosine is selected from N6-methyladenosine, 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N6-methyladenosine One or more of the base adenosine.
  • the chemically modified guanosine is selected from 2-fluoro-2-deoxyguanosine, N7-methyl-guanosine and 2-fluoro-2-deoxy-N7- One or more of methyl-guanosine.
  • various types of chemical modifications preferably include the aforementioned A1-A9, B1-B9, C1-C9, D1-D9, E1-E8, F1- The cases described in F9, G1 to G11 and H1 to H17.
  • the present invention also includes other modification combinations, and the above modifications listed in the examples of the present invention are for illustration only and are not intended to limit the protection scope of the present invention; the present invention also includes other modification combinations.
  • the modified nucleic acid is preferably mRNA, and the modified nucleic acid preferably includes the 5'UTR, 3'UTR and 5'cap structure of the Kozak sequence.
  • the modified nucleic acid in order to facilitate collection and purification, preferably further includes a poly A tail.
  • the present invention does not specifically limit the 5'UTR, 3'UTR, 5'cap structure and poly-A tail including the Kozak sequence, and the above-mentioned structures known in the art can be used; in the present invention, the modified nucleic acid is preferably It also includes a promoter sequence, such as one of T7 promoter, T3 promoter and SP6 promoter; in the present invention, the length of the poly A tail is preferably between 20 and 500 bp.
  • the related sequences involved in the specific implementation process of the present invention are shown in Table 1.
  • the mRNA encoding the viral spike protein is preferably the mRNA of the spike protein (S) of the novel coronavirus 2019-nCov as an example; the mRNA encoding the protease in the organism is luciferase (Luc)
  • the mRNA of the protein hormone is taken as an example; the mRNA of the protein hormone is exemplified by the mRNA of erythropoietin (EPO) and the mRNA encoding human transforming growth factor TGF ⁇ 3; it should be noted that the above-mentioned specific chemically modified nucleic acids of the present invention are only examples The description does not constitute a limitation on the protection scope of the present invention.
  • the present invention provides the use of the modified nucleic acid in the preparation of a disease diagnostic agent and/or a therapeutic agent.
  • the application of the modified nucleic acid is determined according to the specific sequence of the modified nucleic acid.
  • the modified nucleic acid has the following advantages: compared with the natural nucleic acid, the modified nucleic acid improves the expression rate, half-life and/or protein concentration, optimizes the protein localization, and can reduce the natural immune response, avoid degradation pathways in organisms.
  • the modified nucleic acid when the modified nucleic acid is mRNA encoding a virus-related protein, the application of the modified nucleic acid in preparing a vaccine is also provided; the expression efficiency of the modified nucleic acid is higher.
  • the invention can enhance the stability of the mRNA and increase the expression of the protein (antigen) by chemically modifying the mRNA of the base encoding the virus-related protein, and the antigen with high expression can better realize the immune response of the vaccine.
  • the present invention also provides a pharmaceutical formulation comprising the modified nucleic acid and an excipient.
  • the excipient is preferably selected from one of physiological saline, citrate buffer and citrate-physiological saline buffer.
  • the pH value of the citrate buffer is preferably 6.35-6.45, more preferably 6.4; the concentration of citric acid in the citrate buffer is preferably 0.08-0.12mol/L, more preferably 0.10mol /L.
  • the citric acid-physiological saline buffer preferably dissolves citric acid with physiological saline as a solvent, and the concentration of citric acid in the citric acid-physiological saline buffer is preferably 0.08-0.12 mol/L, more preferably is 0.10mol/L.
  • the present invention does not limit the concentration of the modified nucleic acid in the pharmaceutical preparation.
  • the excipient can improve the stability of the modified nucleic acid in terms of concentration, purity and pH value, and is convenient for storage.
  • the drug is administered orally or injected according to the type of the specific drug preparation to achieve its therapeutic effect; the dosage of the drug preparation is determined according to the specific drug and the symptoms of the patient.
  • PCR products include at least
  • A) a promoter sequence (any one of T7 promoter, T3 promoter or SP6 promoter);
  • Reaction volume 50 ⁇ L (reaction volume for a single tube, multiple tubes are simultaneously reacted at one time), and the specific reaction system is shown in Table 2.
  • dNTPs 1 ⁇ l Mg 2+ 1 ⁇ l 10 ⁇ mol/L primer F 1.5 ⁇ l 10 ⁇ mol/L primer R 1.5 ⁇ l 1ng/ ⁇ l synthetic EPO or Luc plasmid template 1 ⁇ l water 18 ⁇ l
  • the reaction procedure was as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10s, annealing at 60°C for 5s, extension at 72°C for 4 min, a total of 34 cycles; and final extension at 72°C for 10 min.
  • reaction solution was combined in a 1.5ml Tube. Take 10 ⁇ l for DNA agarose gel electrophoresis detection to determine the success of the reaction (agarose gel electrophoresis detection conditions: 1.5% agarose, 5V/min, 40min).
  • the plasmid contains the following elements:
  • E) may contain polyadenylic acid sequence (polyA);
  • Millipore 30Kd ultrafiltration tubes concentrate Luc or EPO DNA template.
  • the Luc or EPO DNA template was purified by FPLC, and the concentration of the purified template and the ratio of 260/280 and 260/230 were detected by NanoDrop; the template with a 260/280 ratio ranging from 1.78 to 1.82 was selected for subsequent operations.
  • the template after FPLC purification was concentrated by Millipore 30Kd ultrafiltration tube, and eluted with RNase-free water to dissolve.
  • the concentration of template after ultrafiltration and the ratios of 260/280 and 260/230 were detected by NanoDrop.
  • Reaction volume 1600 ⁇ l (placed in a 2ml RNase-free Tube, which is the reaction volume of a single tube, and multiple tubes are simultaneously reacted at one time).
  • * is composed of uridine, cytosine, adenosine, guanosine or chemically modified nucleosides.
  • Cap analogue# is a commercially available cap structure analog
  • the chemically modified nucleosides include the following:
  • N6-methyladenosine, 2-fluoro-2-deoxyadenosine or 2-fluoro-2-deoxy-N6-methyladenosine are examples of N6-methyladenosine, 2-fluoro-2-deoxyadenosine or 2-fluoro-2-deoxy-N6-methyladenosine.
  • A1 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxyadenosine;
  • A2 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxycytidine;
  • A3 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxyguanosine;
  • A4 is the combined modification of 2-fluoro-2-deoxyuridine and 5-methylcytidine
  • A5 is the combined modification of 2-fluoro-2-deoxyuridine and N7-methyl-guanosine
  • A6 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-5-methylcytidine;
  • A7 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • A8 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-N4-acetylcytidine;
  • A9 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • B1 is the combined modification of pseudouridine and 2-fluoro-2-deoxyadenosine
  • B2 is the combined modification of pseudouridine and 2-fluoro-2-deoxycytidine
  • B3 is the combined modification of pseudouridine and 2-fluoro-2-deoxyguanosine
  • B4 is the combined modification of pseudouridine and 5-methylcytidine
  • B5 is the combined modification of pseudouridine and N7-methyl-guanosine
  • C1 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxyadenosine;
  • C2 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxycytidine;
  • C3 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxyguanosine;
  • C4 is the combined modification of N1-methyl-pseudouridine and 5-methylcytidine;
  • C5 is the combined modification of N1-methyl-pseudouridine and N7-methyl-guanosine;
  • C6 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methylcytidine;
  • C7 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • C8 is the combined modification of N1-methyl-pseudouridine 2-fluoro-2-deoxy-N4-acetylcytidine;
  • C9 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • D1 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxyadenosine
  • D2 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxycytidine
  • D3 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxyguanosine
  • D4 is the combined modification of 5-methoxyuridine and 5-methylcytidine
  • D5 is the combined modification of 5-methoxyuridine and N7-methyl-guanosine
  • D6 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-5-methylcytidine
  • D7 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • D8 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-N4-acetylcytidine;
  • D9 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-N6-methyladenosine
  • E1 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxyadenosine;
  • E2 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxyguanosine;
  • E3 is the combined modification of N4-acetylcytidine and N7-methyl-guanosine;
  • E4 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-pseudouridine;
  • E5 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • E6 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • E7 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-5-methoxyuridine;
  • E8 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • F1 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxycytidine
  • F2 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxyguanosine
  • F3 is the combined modification of N6-methyladenosine and 5-methylcytidine
  • F4 is the combined modification of N6-methyladenosine and N7-methyl-guanosine
  • F5 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-5-methylcytidine
  • F6 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-pseudouridine;
  • F7 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • F8 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • F9 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-5-methoxyuridine;
  • F10 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
  • G1 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-pseudouridine;
  • G2 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • G3 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • G4 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-5-methoxyuridine;
  • G5 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • G6 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-5-methylcytidine;
  • G7 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-pseudouridine;
  • G8 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • G9 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-5-methoxyuridine;
  • G10 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
  • G11 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • H1 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-5-methylcytidine;
  • H2 is a combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-pseudouridine;
  • H3 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • H4 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • H5 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-5-methoxyuridine;
  • H6 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
  • H7 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-pseudouridine
  • H8 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • H9 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
  • H10 is the combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-5-methoxyuridine
  • H11 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • H12 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-5-methylcytidine;
  • H13 is a combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-pseudouridine;
  • H14 is a combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
  • H15 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-5-methoxyuridine;
  • H16 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
  • H17 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-N6-methyladenosine;
  • DNA template obtained by PCR method or linear plasmid method does not contain polyadenylic acid sequence (polyA)
  • a tailing reaction is required here. The specific steps are as follows:
  • the recovered mRNA concentration and the ratio of 260/280 and 260/230 were detected by NanoDrop.
  • the mRNA from the previous step was subjected to FPLC purification.
  • Millipore 30Kd ultrafiltration FPLC-purified mRNA Millipore 30Kd ultrafiltration FPLC-purified mRNA.
  • the dissolved mRNA concentration and the ratio of 260/280 and 260/230 were detected by NanoDrop.
  • the evaluation standard is the modified mRNA immunoprecipitation test (RIP analysis) to detect Toll-like receptors TRL3, TRL7, TRL8 and RIG-1 and mRNA
  • RIP analysis modified mRNA immunoprecipitation test
  • Preparation of transfection system take 200 ⁇ l opti-MEM, add 10 ⁇ g test substance (concentration 2 ⁇ g/ ⁇ l, 5 ⁇ l) or negative control GFP-mRNA, mix by pipetting gently, then add 60 ⁇ l PEI (concentration 1mg/ml) , immediately placed on a vortex shaker for 10 times, 1 s each time, mixed well, and let stand for 10 min.
  • the prepared transfection system is directly and evenly dropped into the cultured cells, and then shaken up and down, so that the transfection system is evenly distributed on the cells. 6h after transfection, the medium was changed, the old medium was aspirated, and each well was replaced with 2 ml of fresh medium (90% DMEM+10% FBS). Harvest 30-36 h after transfection.
  • TNF ⁇ and IL-8 levels in human PBMCs were studied using cells obtained after transfection of various mRNAs.
  • Enzyme-linked immunosorbent assay (elisa) was performed with human IL-8 and TNFa kit (RayBio).
  • RNA samples obtained by RIP were used for cDNA synthesis using the takara reverse transcription kit, and quantitative PCR experiments were performed using the BioRad SYBR GREEN kit to calculate the copy number relationship between unmodified mRNA, single type of modified mRNA and multiple types of modified mRNA samples.
  • the single-modified or multi-modified mRNA encoding EPO of the present invention its immunogenicity is detected, and the evaluation standard is the level of TNF ⁇ and IL-8 in serum after intramuscular injection of the modified mRNA into mice.
  • balbc mice purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • Various types of modified EPO mRNA were injected intramuscularly into balb/c mice, and the injection dose of each mouse was 100 ⁇ g.
  • the orbital blood was collected from the mice, and the serum was separated.
  • Enzyme-linked immunosorbent assay (elisa) was performed with mouse IL-8 and TNFa kit (RayBio).
  • the modified luciferase (Luc) mRNA prepared in the above example was directly introduced into the lungs of mice through a high-pressure spray device for intratracheal administration, and the in vivo bioluminescence signal characterized the expression intensity and expression time of the modified mRNA in vivo.
  • mice were anesthetized with sodium pentobarbital, and the abdomen was fixed on the injection platform so that the angle of the upper teeth was 45°.
  • a small tongue depressor was used to open the lower jaw of the mouse, and the tongue was pulled with blunt forceps.
  • a high pressure nebulizer needle was inserted into the trachea, 30 ⁇ l of Luc mRNA (1 ⁇ g/ ⁇ l) solution was administered continuously, and the mice were removed.
  • the D-luciferin substrate was dissolved in physiological saline at a concentration of 15 mg/ml, and 100 ⁇ l of this solution was injected into mice via tail vein. After 10 min, the signal intensity in the lungs was quantitatively analyzed using the IVIS small animal imaging system.
  • the expression efficiency of the mRNA of chemically modified EPO prepared in the above examples was evaluated by detecting the EPO protein content and hematocrit value in the blood.
  • Standard dilution Dilute the EPO standard with coating buffer to ST1: 3857ng/ml, ST2: 1928ng/ml, ST3: 964ng/ml, ST4: 482ng/ml, ST5: 241ng/ml, ST6: 121ng/ml ml, with coating buffer as negative control.
  • washing solution (1 ⁇ ): Dilute 50 ⁇ Washing buffer 50 times with sterile water for injection, and mix well.
  • wash the plate Take out the coated 96-well plate from the refrigerator at 4°C, pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add 300 ⁇ l of washing solution (1 ⁇ ) to each well, and let it stand for a while. Set for 30s, pour the liquid in the wells into the waste liquid tank, and repeat the plate washing 4 times to pat the wells dry.
  • Blocking Add Blocking buffer, 250 ⁇ l/well, seal with sealing film, and block at room temperature for 2h.
  • wash the plate pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 ⁇ l of washing solution (1 ⁇ ) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
  • wash the plate pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 ⁇ l of washing solution (1 ⁇ ) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
  • wash the plate pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 ⁇ l of washing solution (1 ⁇ ) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
  • the prepared EPO mRNA was dissolved in RNase-free water, physiological saline, citrate buffer and citrate-physiological saline buffer at an initial concentration of 2224ng/ ⁇ l. Packaged in a 1ml neutral borosilicate bottle, a halogenated butyl rubber stopper 11-B for injection, and an aluminum cap crimp. Investigate the stability of mRNA concentration (content), purity and pH of its main components under the conditions of 2 ⁇ 8°C ⁇ strong light (4500 ⁇ 500Lx).
  • the purity of mRNA dissolved in water decreased from 99.3% to 93.2%, the overall decrease was 6.14%, the concentration decreased from 2224ng/ ⁇ l to 1900ng/ ⁇ l, the overall decrease was 14.57%, and the pH value increased by 0.9.
  • the purity of mRNA dissolved in normal saline decreased from 99.2% to 94.0%, the overall decrease was 5.24%, the concentration decreased from 2224ng/ ⁇ l to 1924ng/ ⁇ l, the overall decrease was 13.49%, and the pH value increased by 0.7.
  • the purity of mRNA dissolved in citrate buffer decreased from 99.3% to 95.1%, the overall decrease was 4.23%, the concentration decreased from 2224ng/ ⁇ l to 1947ng/ ⁇ l, the overall decrease was 12.46%, and the pH value increased by 0.8.
  • the purity of mRNA dissolved in citrate saline buffer decreased from 99.3% to 96.7%, the overall decrease was 2.62%, the concentration decreased from 2224ng/ ⁇ l to 2048ng/ ⁇ l, the overall decrease was 7.91%, and the pH value increased 0.3.
  • the modified luciferase (Luc) mRNA prepared in the above example was directly introduced into the lungs of mice through a high-pressure spray device for intratracheal administration, and the in vivo bioluminescence signal characterized the expression intensity and expression time of the modified mRNA in vivo.
  • mice purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • mice purchasedd from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • the mouse jaws were opened and the oropharynx was exposed by pulling the tongue to one side with blunt clamps, a high pressure nebulizer needle was inserted into the trachea, 30 ⁇ l of Luc mRNA (1 ⁇ g/ ⁇ l) solution was administered continuously, and the mice were removed.
  • the expression of luciferase in mice was detected after 24h.
  • the D-luciferin substrate was dissolved in physiological saline at a concentration of 15 mg/ml, and 100 ⁇ l of this solution was injected into mice via tail vein. After 10 min, use the IVIS small animal imaging system to quantitatively analyze the signal intensity in the lungs.
  • mice 100 ⁇ g unmodified and 100 ⁇ g modified S protein mRNA were injected into mice by intramuscular injection. After 4 weeks, orbital blood was collected from mice, and ELISA experiments were performed to detect antibody titers.
  • Standard dilution Dilute the S protein standard by 200ng/ml with coating buffer and use the coating buffer as a negative control.
  • washing solution (1 ⁇ ): Dilute 50 ⁇ Washing buffer 50 times with sterile water for injection, and mix well.
  • wash the plate Take out the coated 96-well plate from the refrigerator at 4°C, pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add 300 ⁇ l of washing solution (1 ⁇ ) to each well, and let it stand for a while. Set for 30s, pour the liquid in the wells into the waste liquid tank, and repeat the plate washing 4 times to pat the wells dry.
  • Blocking Add Blocking buffer, 250 ⁇ l/well, seal with sealing film, and block at room temperature for 2h.
  • wash the plate pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 ⁇ l of washing solution (1 ⁇ ) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
  • wash the plate pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 ⁇ l of washing solution (1 ⁇ ) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
  • wash the plate pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 ⁇ l of washing solution (1 ⁇ ) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
  • the mRNA of 100 ⁇ g unmodified and 100 ⁇ g modified RBD protein was injected into mice by intramuscular injection. After 24 hours, the orbital blood was collected from the mice, and the concentration of RBD was detected by ELISA experiment. At the same time, the orbital blood was collected again 2 weeks later to detect the RBD antibody titer.
  • Standard dilution use coating buffer to dilute the RBD protein standard to 400ng/ml, and dilute 6 gradients 10 times in sequence, with coating buffer as negative control.
  • Coating Take a 96-well plate and add 7 concentration gradients of standard solution, mouse serum, and negative control in sequence, 100 ⁇ l/well, and 2 wells in parallel. Block overnight at 2-8°C.
  • washing solution (1 ⁇ ): Dilute 50 ⁇ Washing buffer 50 times with sterile water for injection, and mix well.
  • wash the plate Take out the coated 96-well plate from the refrigerator at 4°C, pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add 300 ⁇ l of washing solution (1 ⁇ ) to each well, and let it stand for a while. Set for 30s, pour the liquid in the wells into the waste liquid tank, and repeat the plate washing 4 times to pat the wells dry.
  • Blocking Add Blocking buffer, 250 ⁇ l/well, seal with sealing film, and block at room temperature for 2h.
  • wash the plate pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add 300 ⁇ l of washing solution (1 ⁇ ) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid tank , repeat the plate washing 4 times and pat the wells dry.
  • wash the plate pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 ⁇ l of washing solution (1 ⁇ ) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
  • wash the plate pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 ⁇ l of washing solution (1 ⁇ ) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
  • Elisa detects RBD antibody titers
  • the method for determining the antibody titer of S protein in Example 8 is the same except that the standard substance is RBD protein.
  • 293 cells were transformed with 100 ⁇ g of unmodified and 100 ⁇ g of modified mRNA. After 24 hours, the cell pellet was collected, the cells were lysed, and the lysate was subjected to ELISA assay to detect the concentration of TGF ⁇ 3 protein.
  • Standard dilution use coating buffer to dilute the TGF ⁇ protein standard to 1 ⁇ g/ml, and dilute 6 gradients 10-fold in sequence.
  • the coating buffer is used as a negative control.
  • Coating Take a 96-well plate and add 7 concentration gradients of standard solution, cell lysate, and negative control in sequence, 100 ⁇ l/well, 2 wells in parallel. Block overnight at 2-8°C.
  • washing solution (1 ⁇ ): Dilute 50 ⁇ Washing buffer 50 times with sterile water for injection, and mix well.
  • wash the plate Take out the coated 96-well plate from the refrigerator at 4°C, pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add 300 ⁇ l of washing solution (1 ⁇ ) to each well, and let it stand for a while. Set for 30s, pour the liquid in the wells into the waste liquid tank, and repeat the plate washing 4 times to pat the wells dry.
  • Blocking Add Blocking buffer, 250 ⁇ l/well, seal with sealing film, and block at room temperature for 2h.
  • wash the plate pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 ⁇ l of washing solution (1 ⁇ ) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
  • wash the plate pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 ⁇ l of washing solution (1 ⁇ ) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
  • wash the plate pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 ⁇ l of washing solution (1 ⁇ ) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
  • the experimental results are shown in Figure 88.
  • the chemically modified bases shown in the present invention can significantly increase the expression efficiency of TGF ⁇ 3 in cells.

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Abstract

The present application relates to the technical field of nucleic acid modification, and provides a modified nucleic acid and an application thereof. The modified nucleic acid comprises uridine, cytidine, adenosine, guanosine, and chemically modified nucleoside. The chemically modified nucleoside comprises one or more of chemically modified uridine, chemically modified cytidine, chemically modified adenosine, and chemically modified guanosine. The modified nucleic acid has high stability, low immunogenicity, and long half-life in vivo; the provided modified nucleic acid can be applied to diagnosis and treatment of diseases as a diagnostic or therapeutic agent, and overcomes the disadvantages such as low stability, high immunogenicity, short half-life in vivo, repeated administration within a short time period, and high cost compared with existing nucleic acids in a natural state; and the application cost is reduced while the effect of a nucleic acid drug is enhanced.

Description

一种修饰的核酸及其应用A modified nucleic acid and its application
本申请要求于2020年12月10日提交中国专利局、申请号为202011457004.4、发明名称为“一种修饰的核酸及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application with the application number 202011457004.4 and the invention titled "A Modified Nucleic Acid and Its Application" filed with the China Patent Office on December 10, 2020, the entire contents of which are incorporated herein by reference middle.
技术领域technical field
本发明属于核酸修饰技术领域，尤其涉及一种修饰的核酸及其应用。The invention belongs to the technical field of nucleic acid modification, and in particular relates to a modified nucleic acid and its application.
背景技术Background technique
早在20世纪70年代，科学家们就在RNA中发现了m6A修饰，但由于技术制约其功能一直未能被很好的揭示。直到2012年，科学家们的研究表明，m6A修饰和mRNA的稳定性、剪接加工、翻译以及microRNA的加工有关。此外，m6A还和干细胞命运、生物节律相关，可以促使干细胞从自我更新状态转向细胞分化，研究人员发现，甲基化会缩短mRNA的半衰期，减少其丰度。可以说，m6A修饰几乎影响RNA代谢的每个步骤。As early as the 1970s, scientists discovered m6A modification in RNA, but its function has not been well revealed due to technical constraints. Until 2012, scientists' studies showed that m6A modification is related to mRNA stability, splicing processing, translation, and microRNA processing. In addition, m6A is also related to stem cell fate and biological rhythm, and can promote stem cells from a state of self-renewal to cell differentiation. The researchers found that methylation shortens the half-life of mRNA and reduces its abundance. It can be said that m6A modification affects almost every step of RNA metabolism.
mRNA中m6A修饰研究虽然已经取得了实质性进展，但仍然存在一些技术挑战和基础科学问题。第一，目前在转录组范围内检测m6A的方法主要依赖于m6A抗体富集，与其质量密切相关，抗体选择不当将造成假阳性；第二，MeRIP-Seq(RNA甲基化测序)方法只能将m6A残基定位在100～200nt(nucleotide，nt指核苷酸)的转录本区域中，无法在全转录组水平上鉴定m6A的精确位置；第三，其它RNA结合蛋白免疫沉淀方法，对于一些小样本或珍贵样品不适用；第四，为什么m6A甲基化酶只对一些mRNA起作用而不是全部，是否还存在其它甲基化相关酶以及这些酶之间是如何协调作用的？第五，其它RNA修饰与m6A之间是否存在联系，它们是否一起调节某种转录物的生物学过程？这些问题仍有待进一步研究。Although substantial progress has been made in the study of m6A modification in mRNA, there are still some technical challenges and basic scientific problems. First, the current methods for detecting m6A in the transcriptome range mainly rely on the enrichment of m6A antibodies, which are closely related to their quality, and improper selection of antibodies will cause false positives; second, the MeRIP-Seq (RNA methylation sequencing) method can only The m6A residues are located in the 100-200nt (nucleotide, nt refers to nucleotide) transcript region, and the precise location of m6A cannot be identified at the whole transcriptome level; third, other RNA-binding protein immunoprecipitation methods, for some Not suitable for small samples or precious samples; Fourth, why does m6A methylase only work on some mRNAs but not all, are there other methylation-related enzymes and how are these enzymes coordinated? Fifth, are there links between other RNA modifications and m6A, and do they together regulate the biological process of a certain transcript? These issues remain for further study.
目前发现的RNA的修饰主要包括以下几种：The currently discovered modifications of RNA mainly include the following:
2'-O-甲基化(Nm)：关于5'帽子的2'-O-甲基化修饰(Nm)，一般认为5'帽子可以促使mRNA和核糖体的结合，能有效地封闭RNA5'末端，以保护mRNA免疫5'核酸外切酶的降解，增强mRNA的稳定性。此外，5'帽子还参加mRNA前体的剪接，参与mRNA3'末端多聚腺苷酸化，mRNA从核中到细胞质中的运输也需要5'帽子的参与。1998年，Wei等发现帽结合蛋白(capbindingprotein，CBP)可以与ploy(A)绑定蛋白(PABP)相互作用，拉近了5'帽子与ploy(A)尾的距离，形成的环状结构可以加强帽结构与CBP的亲和力，加快核糖体的循环，从而提高翻译效率(Wei,C.-C.,Balasta,M.L.,Ren,J.&Goss,D.J.(1998).Wheat germ poly(A)binding protein enhances the binding affinity of eukaryotic initiation factor 4F and(iso)4F for cap analogues.Biochemistry 37,1910-1916.)。2010年，Daffis等发现病毒RNA5'帽子的2'-O-甲基化修饰可以使其逃脱宿主的抗病毒应答，细胞质RNA5'帽子的2'-O-甲基化修饰可能是宿主区分自身RNA和外来RNA的一个重要标识(Daffis,S.,Szretter,K.J.,Schriewer,J.&other authors(2010).2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members.Nature 468,452-456.)。哺乳动物细胞对5'帽子没有2'-O-甲基化修饰的病毒具有天然的免疫，因为IFIT1(interferon-induced protein with tetratricopetide repeats 1，干扰素诱导的四肽重复蛋白1)可以与这类病毒mRNA结合，使其不能被翻译。2'-O-methylation (Nm): Regarding the 2'-O-methylation modification (Nm) of the 5' cap, it is generally believed that the 5' cap can promote the binding of mRNA and ribosome, and can effectively block RNA 5' end to protect mRNA from 5' exonuclease degradation and enhance mRNA stability. In addition, the 5' cap also participates in the splicing of mRNA precursors, participates in the polyadenylation of the 3' end of mRNA, and the transport of mRNA from the nucleus to the cytoplasm also requires the participation of the 5' cap. In 1998, Wei et al. found that cap binding protein (CBP) can interact with ploy(A) binding protein (PABP), shortening the distance between the 5' cap and the tail of ploy(A), and the formed ring structure can Strengthen the affinity of the cap structure and CBP, speed up the ribosome recycling, thereby improving the translation efficiency (Wei, C.-C., Balasta, M.L., Ren, J. & Goss, D.J. (1998). Wheat germ poly(A) binding protein enhances the binding affinity of eukaryotic initiation factor 4F and (iso)4F for cap analogies. Biochemistry 37, 1910-1916.). In 2010, Daffis et al. found that the 2'-O-methylation modification of the 5' cap of viral RNA can allow it to escape the host's antiviral response, and the 2'-O-methylation modification of the 5' cap of cytoplasmic RNA may be the host to distinguish its own RNA. and an important marker of foreign RNA (Daffis, S., Szretter, K.J., Schriewer, J. & other authors (2010). 2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature 468, 452-456. ). Mammalian cells are innately immune to viruses without 2'-O-methylation in their 5' caps, because IFIT1 (interferon-induced protein with tetratricopetide repeats 1, interferon-induced tetrapeptide repeats 1) can interact with such Viral mRNA binds so that it cannot be translated.
假尿嘧啶(ψ)：假尿嘧啶修饰是最丰富的RNA修饰，一般由尿苷的异构化产生，已有的研究表明，mRNA的假尿嘧啶化修饰主要有三个功能：改变密码子、增强转录本稳定性和应激反应应答。mRNA假尿嘧啶化修饰的过程是由假尿嘧啶合成酶(pseudouridine synthases，PUS)进行催化，让尿嘧啶核苷酸(U)化学结构发生改变，形成假尿嘧啶核苷酸。之前研究已在tRNA、rRNA、snRNA中发现了大量的假尿嘧啶，最近的研究证实假尿嘧啶同样存在于mRNA中。Pseudouracil (ψ): Pseudouracil modification is the most abundant RNA modification, generally produced by the isomerization of uridine. Previous studies have shown that pseudouracil modification of mRNA has three main functions: changing codons, Enhances transcript stability and stress response. The process of mRNA pseudouracil modification is catalyzed by pseudouracil synthases (PUS), which changes the chemical structure of uracil nucleotides (U) to form pseudouracil nucleotides. Previous studies have found a large number of pseudouracils in tRNA, rRNA, and snRNA, and recent studies have confirmed that pseudouracils also exist in mRNA.
2011年，Karijolich等发现，mRNA上的假尿嘧啶化修饰可以改变密码子。将酵母的密码子中的尿嘧啶(U)替换为假尿嘧啶，并不影响密码子对应编码氨基酸的功能(Karijolich,J.&Yu,Y.-T.(2011).Converting nonsense codons into sense codons by targeted pseudouridylation.Nature 474,395-398.)；2014年，Schwartz等发现，酵母发生热休克时，会由Pus7p(一种蛋白)额外引入超过200个假尿嘧啶修饰位点，如果敲除PUS7基因，则那些含有新引入修饰的mRNA会减少，这说明着假尿嘧啶修饰可能会增强转录本稳定性(Schwartz,S.,Bernstein,D.A.,Mumbach,M.R.&other authors(2014).Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.Cell 159,148-162.)。In 2011, Karijolich et al. found that pseudouracillation on mRNA can change codons. Replacing uracil (U) in yeast codons with pseudouracil does not affect the function of codons corresponding to encoded amino acids (Karijolich, J. & Yu, Y.-T. (2011). Converting nonsense codons into sense codons by targeted pseudouridylation.Nature 474,395-398.); In 2014, Schwartz et al. found that Pus7p (a protein) introduces more than 200 additional pseudouracil modification sites when yeast undergoes heat shock. If the PUS7 gene is knocked out, Then those mRNAs with newly introduced modifications were reduced, suggesting that pseudouracil modifications may enhance transcript stability (Schwartz, S., Bernstein, D.A., Mumbach, M.R. & other authors (2014). Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA. Cell 159, 148-162.).
虽然以上三种修饰已被广泛应用于mRNA药物的生产过程中，但关于mRNA修饰点位的探究才刚刚开始，检测技术的局限性仍有待突破。ψ-seq和RiboMeth-seq(两种测序技术)对假尿嘧啶化修饰和2'-O-核糖甲基化修饰位点的定位可以达到单核苷酸的精度，但对m6A位点的定位精度还不够高。Although the above three modifications have been widely used in the production process of mRNA drugs, the exploration of mRNA modification sites has just begun, and the limitations of detection technology still need to be overcome. ψ-seq and RiboMeth-seq (two sequencing technologies) can achieve single-nucleotide accuracy for the mapping of pseudouracillation and 2'-O-ribose methylation sites, but the mapping of m6A sites Accuracy is not high enough.
此外，还有很多RNA修饰缺乏相应的技术手段去深入研究。因此，在技术方面还需要有更多的“NGS+”型(传统检测手段与高通量测序相结合)的高通量技术产生。最近，纳米孔技术是一种新颖的单分子方法，已显示对m6A的单碱基分辨率检测，科学家认为，这种单分子方法可能会成为一种新颖的范例，可以同时检测不同的RNA修饰。In addition, there are many RNA modifications that lack corresponding technical means to study in depth. Therefore, in terms of technology, more high-throughput technologies of the "NGS+" type (combination of traditional detection methods and high-throughput sequencing) are needed. Recently, nanopore technology, a novel single-molecule approach, has shown single-base resolution detection of m6A, and scientists believe that this single-molecule approach may become a novel paradigm to simultaneously detect different RNA modifications .
目前，对于RNA修饰以及修饰后的RNA性能变化以及应用尚不清楚。At present, it is not clear about RNA modification and the performance changes and applications of modified RNA.
发明内容SUMMARY OF THE INVENTION
有鉴于此，本发明的目的在于提供一种修饰的核酸及其应用，所述修饰的核酸采用人工合成的方法制备，通过将若干种化学修饰的核苷酸合成获得修饰的核酸，所述修饰的核酸稳定性高、免疫原性低、体内半衰期长；具有广泛的应用。In view of this, the object of the present invention is to provide a modified nucleic acid and its application, the modified nucleic acid is prepared by artificial synthesis, and the modified nucleic acid is obtained by synthesizing several chemically modified nucleotides, and the modified nucleic acid is obtained by synthesizing several chemically modified nucleotides. The nucleic acid has high stability, low immunogenicity and long in vivo half-life; it has a wide range of applications.
为了实现上述发明目的，本发明提供了以下技术方案：In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
本发明提供了一种修饰的核酸，包括尿嘧啶核苷、胞嘧啶核苷、腺嘌呤核苷、鸟嘌呤核苷和化学修饰的核苷；所述化学修饰的核苷包括化学修饰的尿嘧啶核苷、化学修饰的胞嘧啶核苷、化学修饰的腺嘌呤核苷和化学修饰的鸟嘌呤核苷中的一种或几种。The present invention provides a modified nucleic acid, including uridine, cytosine, adenosine, guanosine and chemically modified nucleosides; the chemically modified nucleosides include chemically modified uracil One or more of nucleosides, chemically modified cytosines, chemically modified adenosines and chemically modified guanosines.
本发明提供了一种修饰的核酸，包括化学修饰的核苷；所述化学修饰的核苷包括化学修饰的尿嘧啶核苷、化学修饰的胞嘧啶核苷、化学修饰的腺嘌呤核苷和化学修饰的鸟嘌呤核苷。The present invention provides a modified nucleic acid, including chemically modified nucleosides; the chemically modified nucleosides include chemically modified uridine nucleosides, chemically modified cytosine nucleosides, chemically modified adenosine and chemically modified nucleosides Modified guanosine.
优选的，修饰的核酸为核糖核酸。Preferably, the modified nucleic acid is ribonucleic acid.
优选的，所述化学修饰的核苷中的化学修饰包括同分异构和/或基团取代；所述基团取代包括甲基取代、甲氧基取代、卤代和N4-乙酰基取代中的一种或几种。Preferably, the chemical modification in the chemically modified nucleosides includes isomerism and/or group substitution; the group substitution includes methyl substitution, methoxy substitution, halogen substitution and N4-acetyl substitution one or more of them.
优选的，所述化学修饰的尿嘧啶核苷选自2-氟-2-脱氧尿苷、假尿苷、N1-甲基-假尿苷、5-甲氧基尿苷、2-氟-2-脱氧-假尿苷、2-氟-2-脱氧-N1-甲基-假尿苷和2-氟-2-脱氧-5-甲氧基尿苷中的一种或几种。Preferably, the chemically modified uridine is selected from 2-fluoro-2-deoxyuridine, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 2-fluoro-2 - one or more of deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methoxyuridine.
优选的，所述化学修饰的胞嘧啶核苷选自N4-乙酰基胞苷、2-氟-2-脱氧胞苷、5-甲基胞苷、2-氟-2-脱氧-5-甲基胞苷和2-氟-2-脱氧-N4-乙酰基胞苷中的一种或几种。Preferably, the chemically modified cytidine is selected from N4-acetylcytidine, 2-fluoro-2-deoxycytidine, 5-methylcytidine, 2-fluoro-2-deoxy-5-methylcytidine One or more of cytidine and 2-fluoro-2-deoxy-N4-acetylcytidine.
优选的，所述化学修饰的腺嘌呤核苷选自N6-甲基腺苷、2-氟-2-脱氧腺苷和2-氟-2-脱氧-N6-甲基腺苷中的一种或几种。Preferably, the chemically modified adenosine is selected from one of N6-methyladenosine, 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N6-methyladenosine or several.
优选的，所述化学修饰的鸟嘌呤核苷选自2-氟-2-脱氧鸟苷、N7-甲基-鸟苷和2-氟-2-脱氧-N7-甲基-鸟苷中的一种或几种。Preferably, the chemically modified guanosine is selected from one of 2-fluoro-2-deoxyguanosine, N7-methyl-guanosine and 2-fluoro-2-deoxy-N7-methyl-guanosine species or several.
优选的，还包括Kozak序列的5'UTR、3'UTR和5'帽子结构。Preferably, the 5'UTR, 3'UTR and 5'cap structure of the Kozak sequence are also included.
优选的，还包括聚A尾。Preferably, a poly-A tail is also included.
优选的，所述修饰的核酸为修饰的mRNA。Preferably, the modified nucleic acid is modified mRNA.
优选的，包括编码病毒刺突蛋白的mRNA。Preferably, mRNA encoding the viral spike protein is included.
优选的，包括编码生物体内蛋白酶和蛋白激素的mRNA。Preferably, mRNAs encoding proteases and protein hormones in organisms are included.
本发明提供了所述修饰的核酸在制备疾病诊断剂和/或治疗剂中的应用。The present invention provides the use of the modified nucleic acid in the preparation of a disease diagnostic agent and/or a therapeutic agent.
本发明提供了所述修饰的核酸在制备疫苗中的应用。The present invention provides the application of the modified nucleic acid in the preparation of vaccine.
本发明提供了一种药物制剂，包括所述修饰的核酸和赋形剂。The present invention provides a pharmaceutical formulation comprising the modified nucleic acid and an excipient.
优选的，所述赋形剂选自生理盐水、柠檬酸缓冲液和柠檬酸-生理盐水缓冲液中的一种。Preferably, the excipient is selected from one of physiological saline, citrate buffer and citrate-physiological saline buffer.
本发明提供的所述修饰的核酸，通过将若干种修饰的核苷酸人工合成获得特定序列的修饰的核酸，所述修饰的核酸稳定性高、免疫原性低、体内半衰期长；本发明提供的修饰的核酸能够作为诊断剂或治疗剂，应用于疾病的诊断和治疗，与现有天然状态的核酸相比，克服了稳定性低、免疫原性高，体内半衰期短，需要短时间内反复给药，成本昂贵等缺点，在增强核酸药物疗效的同时降低了应用成本。The modified nucleic acid provided by the present invention is obtained by artificially synthesizing several modified nucleotides to obtain a modified nucleic acid with a specific sequence, and the modified nucleic acid has high stability, low immunogenicity and long in vivo half-life; the present invention provides The modified nucleic acid can be used as a diagnostic or therapeutic agent for the diagnosis and treatment of diseases. Compared with the existing nucleic acid in its natural state, it overcomes the problems of low stability, high immunogenicity, short half-life in vivo, and the need for repeated cycles in a short time. The disadvantages of drug administration, high cost, etc., reduce the application cost while enhancing the efficacy of nucleic acid drugs.
本发明提供的修饰促红细胞生成素(EPO)的mRNA与TRL3、TRL7、TRL8和RIG-1的结合相对于未修饰的EPO的mRNA显著减少，且TNFα和IL-8水平也显著减少，多种类修饰的mRNA比单种类修饰的mRNA明显更有效。本发明提供的单种类修饰和多种类修饰的mRNA显著减少Toll样受体的结合，从而减少免疫应答，说明修饰的mRNA相较未修饰的mRNA免疫原性低，有利于体内的应用。The binding of the modified erythropoietin (EPO) mRNA provided by the present invention to TRL3, TRL7, TRL8 and RIG-1 is significantly reduced compared to the unmodified EPO mRNA, and the levels of TNFα and IL-8 are also significantly reduced. Modified mRNAs are significantly more efficient than single species of modified mRNAs. The single-type modified and multi-type modified mRNAs provided by the present invention significantly reduce the binding of Toll-like receptors, thereby reducing the immune response, indicating that the modified mRNAs are less immunogenic than unmodified mRNAs, which are beneficial for in vivo applications.
本发明提供的修饰的核酸，例如修饰的促红细胞生成素(EPO)的mRNA、修饰的荧光素酶(Luc)的mRNA，与对应未修饰的mRNA相比，在小鼠体内的表达量更高、表达时间更长、表达更稳定，并且能够发挥相应的作用，其中多种类的修饰的mRNA的效果优于单一种类的修饰。The modified nucleic acids provided by the present invention, such as modified erythropoietin (EPO) mRNA and modified luciferase (Luc) mRNA, have higher expression levels in mice than the corresponding unmodified mRNAs , the expression time is longer, the expression is more stable, and can play a corresponding role, wherein the effect of multiple types of modified mRNA is better than that of a single type of modification.
本发明提供的药物制剂，将所述修饰的核酸与赋形剂混合获得药物制剂，相比单独的修饰的核酸，在修饰的核 酸的纯度、浓度和pH值方面，稳定性更好，利于储存。In the pharmaceutical preparation provided by the present invention, the modified nucleic acid is mixed with excipients to obtain the pharmaceutical preparation. Compared with the modified nucleic acid alone, the modified nucleic acid has better stability in terms of purity, concentration and pH value, and is convenient for storage. .
说明书附图Instruction drawings
图1为单一种类化学修饰mRNA在细胞内与胞内toll样受体TRL3结合水平；Figure 1 shows the binding level of a single chemically modified mRNA to the intracellular toll-like receptor TRL3 in cells;
图2～图9为多种类化学修饰的mRNA在细胞内与胞内toll样受体TRL3结合水平；Figures 2 to 9 show the intracellular binding levels of various chemically modified mRNAs to the intracellular toll-like receptor TRL3;
图10为单一种类化学修饰mRNA在细胞内与胞内toll样受体TRL7结合水平；Figure 10 shows the binding level of a single chemically modified mRNA to the intracellular toll-like receptor TRL7 in cells;
图11～图18为多种类化学修饰的mRNA在细胞内与胞内toll样受体TRL7结合水平；Figures 11 to 18 show the intracellular binding levels of various chemically modified mRNAs to the intracellular toll-like receptor TRL7;
图19为单一种类化学修饰mRNA在细胞内与胞内toll样受体TRL8结合水平；Figure 19 shows the binding level of a single chemically modified mRNA to the intracellular toll-like receptor TRL8 in cells;
图20～27为多种类化学修饰的mRNA在细胞内与胞内toll样受体TRL8结合水平；Figures 20-27 show the binding levels of various chemically modified mRNAs to the intracellular toll-like receptor TRL8 in cells;
图28为单一种类化学修饰mRNA在细胞内与胞内toll样受体RIG-1结合水平；Figure 28 shows the binding level of a single chemically modified mRNA to the intracellular toll-like receptor RIG-1 in cells;
图29～图36为多种类化学修饰的mRNA在细胞内与胞内toll样受体RIG-1结合水平；Figures 29 to 36 are the intracellular binding levels of various chemically modified mRNAs to the intracellular toll-like receptor RIG-1;
图37为单一种类化学修饰mRNA注射小鼠后，小鼠血清中IL-8的含量变化；Figure 37 shows the changes in the content of IL-8 in mouse serum after a single chemically modified mRNA was injected into mice;
[根据细则91更正 18.02.2021]　
图38～图44为多种类化学修饰的mRNA注射小鼠后，小鼠血清中IL-8的含量变化；[Correction 18.02.2021 in accordance with Rule 91]
Figures 38 to 44 show the changes in the content of IL-8 in the serum of mice after injection of various types of chemically modified mRNA into mice;
图45为单一种类化学修饰mRNA注射小鼠后，小鼠血清中TNFα的含量变化；Figure 45 shows the changes in the content of TNFα in mouse serum after a single chemically modified mRNA was injected into mice;
图46～图53为多种类化学修饰的mRNA注射小鼠后，小鼠血清中TNFα的含量变化；Figures 46 to 53 show the changes in the content of TNFα in the serum of mice after injection of various chemically modified mRNAs into mice;
图54为单一种类化学修饰mRNA注射小鼠后，小鼠体内荧光素酶表达情况；Figure 54 shows the expression of luciferase in mice after injection of a single chemically modified mRNA into mice;
图55～图62为多种类化学修饰mRNA注射小鼠后，小鼠体内荧光素酶表达情况；Figures 55 to 62 show the expression of luciferase in mice after various types of chemically modified mRNAs were injected into mice;
图63为单一种类化学修饰mRNA注射小鼠后，小鼠血清中促红细胞生成素(EPO)表达情况；Figure 63 shows the expression of erythropoietin (EPO) in mouse serum after a single chemically modified mRNA was injected into mice;
图64～图71为多种类化学修饰mRNA注射小鼠后，小鼠血清中促红细胞生成素(EPO)的含量；Figures 64 to 71 show the content of erythropoietin (EPO) in the serum of mice after injection of various chemically modified mRNAs into mice;
图72为单一种类化学修饰mRNA注射小鼠后，小鼠的红细胞压积；Figure 72 shows the hematocrit of mice after injection of a single chemically modified mRNA into mice;
图73～图80为多种类化学修饰mRNA注射小鼠后，小鼠的红细胞压积；Figures 73 to 80 show the hematocrit of mice after injection of various chemically modified mRNAs into mice;
图81为不同赋形剂对mRNA药物存储条件下pH的影响；Figure 81 is the effect of different excipients on pH under mRNA drug storage conditions;
图82为不同赋形剂对mRNA药物存储条件下纯度的影响；Figure 82 is the effect of different excipients on the purity of mRNA drug storage conditions;
图83为不同赋形剂对mRNA药物存储条件下浓度的影响；Figure 83 is the effect of different excipients on the concentration of mRNA drug storage conditions;
图84为修饰碱基的掺入比例与荧光素酶(Luc)mRNA表达水平的高低之间的关系；Figure 84 shows the relationship between the incorporation ratio of modified bases and the expression level of luciferase (Luc) mRNA;
图85为部分碱基修饰和全部碱基修饰对新型冠状病毒2019-nCov的刺突蛋白(S)的mRNA表达水平的影响。Figure 85 shows the effects of partial base modifications and all base modifications on the mRNA expression level of the spike protein (S) of the novel coronavirus 2019-nCov.
图86为不同化学修饰策略的新冠病毒RBD mRNA表达蛋白浓度结果；图87为不同化学修饰策略的新冠病毒RBD mRNA表达蛋白抗体滴度结果；Figure 86 shows the results of the new coronavirus RBD mRNA expression protein concentration results for different chemical modification strategies; Figure 87 shows the antibody titer results of the new coronavirus RBD mRNA expression protein for different chemical modification strategies;
图88为不同化学修饰策略的人转化生长因子TGFβ3mRNA表达结果；Figure 88 shows the expression results of human transforming growth factor TGFβ3 mRNA with different chemical modification strategies;
上述附图中，In the above drawings,
A1为2-氟-2-脱氧尿苷和2-氟-2-脱氧腺苷联合修饰；A1 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxyadenosine;
A2为2-氟-2-脱氧尿苷和2-氟-2-脱氧胞苷联合修饰；A2 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxycytidine;
A3为2-氟-2-脱氧尿苷和2-氟-2-脱氧鸟苷联合修饰；A3 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxyguanosine;
A4为2-氟-2-脱氧尿苷和5-甲基胞苷联合修饰；A4 is the combined modification of 2-fluoro-2-deoxyuridine and 5-methylcytidine;
A5为2-氟-2-脱氧尿苷和N7-甲基-鸟苷联合修饰；A5 is the combined modification of 2-fluoro-2-deoxyuridine and N7-methyl-guanosine;
A6为2-氟-2-脱氧尿苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；A6 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-5-methylcytidine;
A7为2-氟-2-脱氧尿苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；A7 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
A8为2-氟-2-脱氧尿苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；A8 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-N4-acetylcytidine;
A9为2-氟-2-脱氧尿苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；A9 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-N6-methyladenosine;
B1为假尿苷和2-氟-2-脱氧腺苷联合修饰；B1 is the combined modification of pseudouridine and 2-fluoro-2-deoxyadenosine;
B2为假尿苷和2-氟-2-脱氧胞苷联合修饰；B2 is the combined modification of pseudouridine and 2-fluoro-2-deoxycytidine;
B3为假尿苷和2-氟-2-脱氧鸟苷联合修饰；B3 is the combined modification of pseudouridine and 2-fluoro-2-deoxyguanosine;
B4为假尿苷和5-甲基胞苷联合修饰；B4 is the combined modification of pseudouridine and 5-methylcytidine;
B5为假尿苷和N7-甲基-鸟苷联合修饰；B5 is the combined modification of pseudouridine and N7-methyl-guanosine;
B6假尿苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；Combined modification of B6 pseudouridine and 2-fluoro-2-deoxy-5-methylcytidine;
B7假尿苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；Combined modification of B7 pseudouridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
B8假尿苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；Combined modification of B8 pseudouridine and 2-fluoro-2-deoxy-N4-acetylcytidine;
B9假尿苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；Combined modification of B9 pseudouridine and 2-fluoro-2-deoxy-N6-methyladenosine;
C1为N1-甲基-假尿苷和2-氟-2-脱氧腺苷联合修饰；C1 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxyadenosine;
C2为N1-甲基-假尿苷和2-氟-2-脱氧胞苷联合修饰；C2 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxycytidine;
C3为N1-甲基-假尿苷和2-氟-2-脱氧鸟苷联合修饰；C3 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxyguanosine;
C4为N1-甲基-假尿苷和5-甲基胞苷联合修饰；C4 is the combined modification of N1-methyl-pseudouridine and 5-methylcytidine;
C5为N1-甲基-假尿苷和N7-甲基-鸟苷联合修饰；C5 is the combined modification of N1-methyl-pseudouridine and N7-methyl-guanosine;
C6为N1-甲基-假尿苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；C6 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methylcytidine;
C7为N1-甲基-假尿苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；C7 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
C8为N1-甲基-假尿苷2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；C8 is the combined modification of N1-methyl-pseudouridine 2-fluoro-2-deoxy-N4-acetylcytidine;
C9为N1-甲基-假尿苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；C9 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxy-N6-methyladenosine;
D1为5-甲氧基尿苷和2-氟-2-脱氧腺苷联合修饰；D1 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxyadenosine;
D2为5-甲氧基尿苷和2-氟-2-脱氧胞苷联合修饰；D2 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxycytidine;
D3为5-甲氧基尿苷和2-氟-2-脱氧鸟苷联合修饰；D3 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxyguanosine;
D4为5-甲氧基尿苷和5-甲基胞苷联合修饰；D4 is the combined modification of 5-methoxyuridine and 5-methylcytidine;
D5为5-甲氧基尿苷和N7-甲基-鸟苷联合修饰；D5 is the combined modification of 5-methoxyuridine and N7-methyl-guanosine;
D6为5-甲氧基尿苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；D6 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-5-methylcytidine;
D7为5-甲氧基尿苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；D7 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
D8为5-甲氧基尿苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；D8 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-N4-acetylcytidine;
D9为5-甲氧基尿苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；D9 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-N6-methyladenosine;
E1为N4-乙酰基胞苷和2-氟-2-脱氧腺苷联合修饰；E1 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxyadenosine;
E2为N4-乙酰基胞苷和2-氟-2-脱氧鸟苷联合修饰；E2 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxyguanosine;
E3为N4-乙酰基胞苷和N7-甲基-鸟苷联合修饰；E3 is the combined modification of N4-acetylcytidine and N7-methyl-guanosine;
E4为N4-乙酰基胞苷和2-氟-2-脱氧-假尿苷联合修饰；E4 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-pseudouridine;
E5为N4-乙酰基胞苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；E5 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
E6为N4-乙酰基胞苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；E6 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
E7为N4-乙酰基胞苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；E7 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-5-methoxyuridine;
E8为N4-乙酰基胞苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；E8 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-N6-methyladenosine;
F1为N6-甲基腺苷和2-氟-2-脱氧胞苷联合修饰；F1 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxycytidine;
F2为N6-甲基腺苷和2-氟-2-脱氧鸟苷联合修饰；F2 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxyguanosine;
F3为N6-甲基腺苷和5-甲基胞苷联合修饰；F3 is the combined modification of N6-methyladenosine and 5-methylcytidine;
F4为N6-甲基腺苷和N7-甲基-鸟苷联合修饰；F4 is the combined modification of N6-methyladenosine and N7-methyl-guanosine;
F5为N6-甲基腺苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；F5 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-5-methylcytidine;
F6为N6-甲基腺苷和2-氟-2-脱氧-假尿苷联合修饰；F6 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-pseudouridine;
F7为N6-甲基腺苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；F7 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
F8为N6-甲基腺苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；F8 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
F9为N6-甲基腺苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；F9 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-5-methoxyuridine;
F10为N6-甲基腺苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；F10 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
G1为2-氟-2-脱氧胞苷和2-氟-2-脱氧-假尿苷联合修饰；G1 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-pseudouridine;
G2为2-氟-2-脱氧胞苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；G2 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
G3为2-氟-2-脱氧胞苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；G3 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
G4为2-氟-2-脱氧胞苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；G4 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-5-methoxyuridine;
G5为2-氟-2-脱氧胞苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；G5 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-N6-methyladenosine;
G6为2-氟-2-脱氧鸟苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；G6 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-5-methylcytidine;
G7为2-氟-2-脱氧鸟苷和2-氟-2-脱氧-假尿苷联合修饰；G7 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-pseudouridine;
G8为2-氟-2-脱氧鸟苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；G8 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
G9为2-氟-2-脱氧鸟苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；G9 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-5-methoxyuridine;
G10为2-氟-2-脱氧鸟苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；G10 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
G11为2-氟-2-脱氧鸟苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；G11 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-N6-methyladenosine;
H1为2-氟-2-脱氧腺苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；H1 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-5-methylcytidine;
H2为2-氟-2-脱氧腺苷和2-氟-2-脱氧-假尿苷联合修饰；H2 is a combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-pseudouridine;
H3为2-氟-2-脱氧腺苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；H3 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
H4为2-氟-2-脱氧腺苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；H4 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
H5为2-氟-2-脱氧腺苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；H5 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-5-methoxyuridine;
H6为2-氟-2-脱氧腺苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；H6 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
H7为5-甲基胞苷和2-氟-2-脱氧-假尿苷联合修饰；H7 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-pseudouridine;
H8为5-甲基胞苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；H8 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
H9为5-甲基胞苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；H9 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
H10为5-甲基胞苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；H10 is the combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-5-methoxyuridine;
H11为5-甲基胞苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；H11 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-N6-methyladenosine;
H12为N7-甲基-鸟苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；H12 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-5-methylcytidine;
H13为N7-甲基-鸟苷和2-氟-2-脱氧-假尿苷联合修饰；H13 is a combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-pseudouridine;
H14为N7-甲基-鸟苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；H14 is a combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
H15为N7-甲基-鸟苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；H15 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-5-methoxyuridine;
H16为N7-甲基-鸟苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；H16 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
H17为N7-甲基-鸟苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰。H17 is a combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-N6-methyladenosine.
具体实施方式Detailed ways
本发明提供了一种修饰的核酸，包括尿嘧啶核苷、胞嘧啶核苷、腺嘌呤核苷、鸟嘌呤核苷和化学修饰的核苷；所述化学修饰的核苷包括化学修饰的尿嘧啶核苷、化学修饰的胞嘧啶核苷、化学修饰的腺嘌呤核苷和化学修饰的鸟嘌呤核苷中的一种或几种。The present invention provides a modified nucleic acid, including uridine, cytosine, adenosine, guanosine and chemically modified nucleosides; the chemically modified nucleosides include chemically modified uracil One or more of nucleosides, chemically modified cytosines, chemically modified adenosines and chemically modified guanosines.
本发明对所述修饰的核酸的具体序列没有特殊限定，任意序列的核酸均可；所述核酸可以为编码生物体内已知的蛋白酶、蛋白激素；也可以为编码生物体内不存在的或未知的蛋白酶；所述核酸还包括编码病毒刺突蛋白的mRNA；所述核酸优选编码疾病相关的蛋白质。The specific sequence of the modified nucleic acid is not particularly limited in the present invention, and any sequence of nucleic acid is acceptable; the nucleic acid may encode known proteases and protein hormones in the organism; it may also encode non-existent or unknown in vivo encoding protease; the nucleic acid also includes mRNA encoding a viral spike protein; the nucleic acid preferably encodes a disease-associated protein.
本发明对所述修饰的核酸中化学修饰的核苷的种类和数量没有特殊限定；在一条修饰的核酸中，可以存在1～4种的修饰的核苷(包括化学修饰的尿嘧啶核苷、化学修饰的胞嘧啶核苷、化学修饰的腺嘌呤核苷和化学修饰的鸟嘌呤核苷)；针对某一种核苷，在一条修饰的核酸中，可以存在不同种类的化学修饰；针对某一种的化学修饰，在一条修饰的核酸中，可以存在不同数量的修饰位点。The present invention does not specifically limit the type and quantity of chemically modified nucleosides in the modified nucleic acid; in a modified nucleic acid, there may be 1 to 4 kinds of modified nucleosides (including chemically modified uridine, chemically modified cytosine nucleosides, chemically modified adenosine nucleosides and chemically modified guanosine nucleosides); for a certain nucleoside, there can be different kinds of chemical modifications in a modified nucleic acid; for a certain nucleoside There can be different numbers of modification sites in a modified nucleic acid.
在本发明中，所述化学修饰的核苷中的化学修饰包括同分异构和/或基团取代；所述基团取代包括甲基取代、甲氧基取代、卤代和N4-乙酰基取代中的一种或几种。在本发明中，所述化学修饰还包括脱氧。In the present invention, the chemical modification in the chemically modified nucleosides includes isomerization and/or group substitution; the group substitution includes methyl substitution, methoxy substitution, halogenation and N4-acetyl group one or more of the substitutions. In the present invention, the chemical modification also includes deoxygenation.
在本发明中，针对尿嘧啶核苷，所述化学修饰的尿嘧啶核苷优选的选自2-氟-2-脱氧尿苷、假尿苷、N1-甲基-假尿苷、5-甲氧基尿苷、2-氟-2-脱氧-假尿苷、2-氟-2-脱氧-N1-甲基-假尿苷和2-氟-2-脱氧-5-甲氧基尿苷中的一种或几种。In the present invention, for uridine, the chemically modified uridine is preferably selected from 2-fluoro-2-deoxyuridine, pseudouridine, N1-methyl-pseuduridine, 5-methyluridine oxyuridine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methoxyuridine one or more of them.
在本发明中，针对胞嘧啶核苷，所述化学修饰的胞嘧啶核苷选自N4-乙酰基胞苷、2-氟-2-脱氧胞苷、5-甲基胞苷、2-氟-2-脱氧-5-甲基胞苷和2-氟-2-脱氧-N4-乙酰基胞苷、中的一种或几种。In the present invention, for cytidine, the chemically modified cytidine is selected from N4-acetylcytidine, 2-fluoro-2-deoxycytidine, 5-methylcytidine, 2-fluoro-cytidine One or more of 2-deoxy-5-methylcytidine and 2-fluoro-2-deoxy-N4-acetylcytidine.
在本发明中，针对腺嘌呤核苷，所述化学修饰的腺嘌呤核苷选自N6-甲基腺苷、2-氟-2-脱氧腺苷和2-氟-2-脱氧-N6-甲基腺苷中的一种或几种。In the present invention, for adenosine, the chemically modified adenosine is selected from N6-methyladenosine, 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N6-methyladenosine One or more of the base adenosine.
在本发明中，针对鸟嘌呤核苷，所述化学修饰的鸟嘌呤核苷选自2-氟-2-脱氧鸟苷、N7-甲基-鸟苷和2-氟-2-脱氧-N7-甲基-鸟苷中的一种或几种。In the present invention, for guanosine, the chemically modified guanosine is selected from 2-fluoro-2-deoxyguanosine, N7-methyl-guanosine and 2-fluoro-2-deoxy-N7- One or more of methyl-guanosine.
在本发明中，针对一条修饰的核酸，在本发明具体实施过程中，多种类的化学修饰优选的包括前述A1～A9、B1～B9、C1～C9、D1～D9、E1～E8、F1～F9、G1～G11和H1～H17中记载的情况。本发明还包括其他修饰组合的情况，本发明实施例中所列举的上述修饰情况，仅为举例说明，不作为本发明保护范围的限定；本发明还包括其他修饰组合的情况。In the present invention, for a modified nucleic acid, in the specific implementation process of the present invention, various types of chemical modifications preferably include the aforementioned A1-A9, B1-B9, C1-C9, D1-D9, E1-E8, F1- The cases described in F9, G1 to G11 and H1 to H17. The present invention also includes other modification combinations, and the above modifications listed in the examples of the present invention are for illustration only and are not intended to limit the protection scope of the present invention; the present invention also includes other modification combinations.
在本发明中，所述修饰的核酸优选为mRNA，所述修饰的核酸优选的包括Kozak序列的5'UTR、3'UTR和5'帽子结构。在本发明中，为了便于收集纯化，所述修饰的核酸优选的还包括聚A尾。本发明对所述包括Kozak序列的5'UTR、3'UTR、5'帽子结构和聚A尾没有特殊限定，采用本领域公知的上述结构即可；本发明中，所述修饰的核酸优选的还包括启动子序列，例如T7启动子、T3启动子和SP6启动子中一种；在本发明中，所述聚A尾的长度优选的在20～500bp之间。本发明在具体实施过程中涉及到的相关序列如表1所示。In the present invention, the modified nucleic acid is preferably mRNA, and the modified nucleic acid preferably includes the 5'UTR, 3'UTR and 5'cap structure of the Kozak sequence. In the present invention, in order to facilitate collection and purification, the modified nucleic acid preferably further includes a poly A tail. The present invention does not specifically limit the 5'UTR, 3'UTR, 5'cap structure and poly-A tail including the Kozak sequence, and the above-mentioned structures known in the art can be used; in the present invention, the modified nucleic acid is preferably It also includes a promoter sequence, such as one of T7 promoter, T3 promoter and SP6 promoter; in the present invention, the length of the poly A tail is preferably between 20 and 500 bp. The related sequences involved in the specific implementation process of the present invention are shown in Table 1.
在本发明中，所述编码病毒刺突蛋白的mRNA优选的以新型冠状病毒2019-nCov的刺突蛋白(S)的mRNA为例；所述编码生物体内蛋白酶的mRNA以荧光素酶(Luc)的mRNA为例；所述蛋白激素的mRNA以促红细胞生成素(EPO)的mRNA、编码人转化生长因子TGFβ3的mRNA为例；需要说明的是，本发明上述具体的化学修饰的核酸仅为举例说明，并不构成对本发明保护范围的限定。In the present invention, the mRNA encoding the viral spike protein is preferably the mRNA of the spike protein (S) of the novel coronavirus 2019-nCov as an example; the mRNA encoding the protease in the organism is luciferase (Luc) The mRNA of the protein hormone is taken as an example; the mRNA of the protein hormone is exemplified by the mRNA of erythropoietin (EPO) and the mRNA encoding human transforming growth factor TGFβ3; it should be noted that the above-mentioned specific chemically modified nucleic acids of the present invention are only examples The description does not constitute a limitation on the protection scope of the present invention.
本发明提供了所述修饰的核酸在制备疾病诊断剂和/或治疗剂中的应用。在本发明中，根据修饰的核酸的具体序 列确定所述修饰的核酸的应用。在本发明中，所述修饰的核酸具有以下优势：相对于天然的核酸，修饰的核酸提高了表达率、半衰期和/或蛋白浓度，优化了蛋白定位，并且能够减低天然的免疫应答反应，避免生物体内的降解途径。The present invention provides the use of the modified nucleic acid in the preparation of a disease diagnostic agent and/or a therapeutic agent. In the present invention, the application of the modified nucleic acid is determined according to the specific sequence of the modified nucleic acid. In the present invention, the modified nucleic acid has the following advantages: compared with the natural nucleic acid, the modified nucleic acid improves the expression rate, half-life and/or protein concentration, optimizes the protein localization, and can reduce the natural immune response, avoid degradation pathways in organisms.
在本发明中，当所述修饰的核酸为编码病毒相关蛋白的mRNA时，还提供了所述修饰的核酸在制备疫苗中的应用；所述修饰的核酸的表达效率更高。本发明通过化学修饰碱基编码病毒相关蛋白的mRNA能够增强mRNA的稳定性、提高蛋白(抗原)表达量，高表达量的抗原能够更好的实现疫苗的免疫反应。In the present invention, when the modified nucleic acid is mRNA encoding a virus-related protein, the application of the modified nucleic acid in preparing a vaccine is also provided; the expression efficiency of the modified nucleic acid is higher. The invention can enhance the stability of the mRNA and increase the expression of the protein (antigen) by chemically modifying the mRNA of the base encoding the virus-related protein, and the antigen with high expression can better realize the immune response of the vaccine.
本发明还提供了一种药物制剂，包括所述修饰的核酸和赋形剂。在本发明中，所述赋形剂优选的选自生理盐水、柠檬酸缓冲液和柠檬酸-生理盐水缓冲液中的一种。在本发明中，所述柠檬酸缓冲液的pH值优选为6.35～6.45，更优选为6.4；所述柠檬酸缓冲液中柠檬酸的浓度优选为0.08～0.12mol/L，更优选为0.10mol/L。在本发明中，所述柠檬酸-生理盐水缓冲液优选的以生理盐水为溶剂溶解柠檬酸，所述柠檬酸-生理盐水缓冲液中柠檬酸的浓度优选为0.08～0.12mol/L，更优选为0.10mol/L。本发明对所述修饰的核酸在所述药物制剂中浓度没有限定。在本发明中，所述赋形剂能够提高所述修饰的核酸在浓度、纯度和pH值方面的稳定，利于储存。The present invention also provides a pharmaceutical formulation comprising the modified nucleic acid and an excipient. In the present invention, the excipient is preferably selected from one of physiological saline, citrate buffer and citrate-physiological saline buffer. In the present invention, the pH value of the citrate buffer is preferably 6.35-6.45, more preferably 6.4; the concentration of citric acid in the citrate buffer is preferably 0.08-0.12mol/L, more preferably 0.10mol /L. In the present invention, the citric acid-physiological saline buffer preferably dissolves citric acid with physiological saline as a solvent, and the concentration of citric acid in the citric acid-physiological saline buffer is preferably 0.08-0.12 mol/L, more preferably is 0.10mol/L. The present invention does not limit the concentration of the modified nucleic acid in the pharmaceutical preparation. In the present invention, the excipient can improve the stability of the modified nucleic acid in terms of concentration, purity and pH value, and is convenient for storage.
在本发明中，根据具体的药物制剂的种类将所述药物进行口服或注射实现其治疗作用；所述药物制剂的使用剂量视具体药物和患者症状确定。In the present invention, the drug is administered orally or injected according to the type of the specific drug preparation to achieve its therapeutic effect; the dosage of the drug preparation is determined according to the specific drug and the symptoms of the patient.
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.
实施例1Example 1
DNA模板制备DNA template preparation
1)PCR法制备荧光素酶(Luc)和促红细胞生成素(EPO)DNA模板，在96孔PCR仪器中进行。1) PCR method to prepare luciferase (Luc) and erythropoietin (EPO) DNA templates, and carry out in a 96-well PCR instrument.
PCR产物中至少包括PCR products include at least
A)一个启动子序列(T7启动子、T3启动子或SP6启动子中任选一个)；A) a promoter sequence (any one of T7 promoter, T3 promoter or SP6 promoter);
B)包含至少一个Kozak序列的5'UTR；B) a 5'UTR comprising at least one Kozak sequence;
C)3'UTR；C) 3'UTR;
D)Luc或EPO编码序列；D) Luc or EPO coding sequence;
E)聚A尾(polyA tail)。E) polyA tail.
本发明中涉及的具体序列如表1所示。The specific sequences involved in the present invention are shown in Table 1.
表1具体序列Table 1 Specific sequence
Figure PCTCN2020138025-appb-000001
Figure PCTCN2020138025-appb-000001
按如下反应体系进行DNA模板的扩增：Amplify the DNA template according to the following reaction system:
反应体积，50μL(为单个管的反应体积，一次同时反应多管)，具体的反应体系见表2。Reaction volume, 50 μL (reaction volume for a single tube, multiple tubes are simultaneously reacted at one time), and the specific reaction system is shown in Table 2.
表2反应体系Table 2 Reaction system
组分component 体积volume
PrimeSTAR Max Premix(2×)PrimeSTAR Max Premix(2×) 25μl25μl
PrimeSTAR Max DNA PolymerasePrimeSTAR Max DNA Polymerase 1μl1μl
dNTPsdNTPs 1μl1μl
Mg 2+ Mg 2+ 1μl1μl
10μmol/L引物F10μmol/L primer F 1.5μl1.5μl
10μmol/L引物R10μmol/L primer R 1.5μl1.5μl
1ng/μl人工合成的EPO或Luc质粒模板1ng/μl synthetic EPO or Luc plasmid template 1μl1μl
水water 18μl18μl
反应程序如下：预变性 98℃ 3min；变性 98℃ 10s，退火 60℃ 5s，延伸 72℃ 4min，共34个循环；最后延伸 72℃ 10min。The reaction procedure was as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10s, annealing at 60°C for 5s, extension at 72°C for 4 min, a total of 34 cycles; and final extension at 72°C for 10 min.
反应结束后，将反应液合并于1.5ml Tube管中。取10μl进行DNA琼脂糖凝胶电泳检测以确定反应成功(琼脂糖凝胶电泳检测条件：1.5％琼脂糖，5V/min，40min)。After the reaction, the reaction solution was combined in a 1.5ml Tube. Take 10 μl for DNA agarose gel electrophoresis detection to determine the success of the reaction (agarose gel electrophoresis detection conditions: 1.5% agarose, 5V/min, 40min).
2)线性质粒作为DNA模板2) Linear plasmid as DNA template
该质粒包含以下元件：The plasmid contains the following elements:
A)一个启动子序列；A) a promoter sequence;
B)包含至少一个Kozak序列的5'UTR；B) a 5'UTR comprising at least one Kozak sequence;
C)Luc或EPO编码序列；C) Luc or EPO coding sequence;
D)3'UTR；D) 3'UTR;
E)可能含有聚腺苷酸序列(polyA)；E) may contain polyadenylic acid sequence (polyA);
F)D)或E)后面有一个限制性内切酶位点；F) D) or E) followed by a restriction endonuclease site;
质粒线性化方法Plasmid linearization method
标准反应体系：Standard reaction system:
Figure PCTCN2020138025-appb-000002
Figure PCTCN2020138025-appb-000002
DNA模板超滤DNA template ultrafiltration
Millipore 30Kd超滤管浓缩Luc或EPO DNA模板。Millipore 30Kd ultrafiltration tubes concentrate Luc or EPO DNA template.
DNA模板FPLC纯化DNA template FPLC purification
FPLC纯化Luc或EPO DNA模板，用NanoDrop检测纯化后模板的浓度，以及260/280、260/230的比值；选择260/280比值范围为1.78～1.82的模板进行后续操作。The Luc or EPO DNA template was purified by FPLC, and the concentration of the purified template and the ratio of 260/280 and 260/230 were detected by NanoDrop; the template with a 260/280 ratio ranging from 1.78 to 1.82 was selected for subsequent operations.
取样进行DNA琼脂糖凝胶电泳检测(1.5％琼脂糖，5V/min，40min)。Samples were taken for DNA agarose gel electrophoresis detection (1.5% agarose, 5V/min, 40min).
FPLC纯化后模板超滤Template ultrafiltration after FPLC purification
Millipore 30Kd超滤管浓缩FPLC纯化后模板，用RNase-free水洗脱溶解。The template after FPLC purification was concentrated by Millipore 30Kd ultrafiltration tube, and eluted with RNase-free water to dissolve.
用NanoDrop检测超滤后模板的浓度，以及260/280、260/230的比值。The concentration of template after ultrafiltration and the ratios of 260/280 and 260/230 were detected by NanoDrop.
最终用RNase-free水稀释至300ng/μl。Final dilution with RNase-free water to 300ng/μl.
mRNA的体外合成In vitro synthesis of mRNA
在恒温反应器中，进行mRNA的体外合成。In a thermostatic reactor, in vitro synthesis of mRNA is performed.
按照如下合成体系进行(反应试剂按照表格从上至下添加)：Carry out the following synthesis system (reagents are added from top to bottom according to the table):
反应体积，1600μl(置于2ml RNase-free Tube管中，为单个管的反应体积，一次同时反应多管)。Reaction volume, 1600μl (placed in a 2ml RNase-free Tube, which is the reaction volume of a single tube, and multiple tubes are simultaneously reacted at one time).
表3化学修饰的mRNA合成体系Table 3 Chemically modified mRNA synthesis systems
Figure PCTCN2020138025-appb-000003
Figure PCTCN2020138025-appb-000003
Figure PCTCN2020138025-appb-000004
Figure PCTCN2020138025-appb-000004
*为尿嘧啶核苷、胞嘧啶核苷、腺嘌呤核苷、鸟嘌呤核苷或化学修饰核苷组成。* is composed of uridine, cytosine, adenosine, guanosine or chemically modified nucleosides.
Cap analogue#为市售帽结构类似物；Cap analogue# is a commercially available cap structure analog;
将恒温器的热盖启动，设置为42℃。Activate the hot lid of the thermostat and set it to 42°C.
点击恒温器反应系统，37℃，6h。Click the thermostat reaction system, 37°C, 6h.
其中化学修饰的核苷包括以下情况：The chemically modified nucleosides include the following:
2-氟-2-脱氧尿苷、假尿苷、N1-甲基-假尿苷、5-甲氧基尿苷、2-氟-2-脱氧-假尿苷、2-氟-2-脱氧-N1-甲基-假尿苷或2-氟-2-脱氧-5-甲氧基尿苷。2-Fluoro-2-deoxyuridine, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy -N1-methyl-pseudouridine or 2-fluoro-2-deoxy-5-methoxyuridine.
N4-乙酰基胞苷、2-氟-2-脱氧胞苷、5-甲基胞苷、2-氟-2-脱氧-5-甲基胞苷或2-氟-2-脱氧-N4-乙酰基胞苷。N4-acetylcytidine, 2-fluoro-2-deoxycytidine, 5-methylcytidine, 2-fluoro-2-deoxy-5-methylcytidine, or 2-fluoro-2-deoxy-N4-acetyl base cytidine.
N6-甲基腺苷、2-氟-2-脱氧腺苷或2-氟-2-脱氧-N6-甲基腺苷。N6-methyladenosine, 2-fluoro-2-deoxyadenosine or 2-fluoro-2-deoxy-N6-methyladenosine.
2-氟-2-脱氧鸟苷、N7-甲基-鸟苷或2-氟-2-脱氧-N7-甲基-鸟苷。2-Fluoro-2-deoxyguanosine, N7-methyl-guanosine or 2-fluoro-2-deoxy-N7-methyl-guanosine.
A1为2-氟-2-脱氧尿苷和2-氟-2-脱氧腺苷联合修饰；A1 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxyadenosine;
A2为2-氟-2-脱氧尿苷和2-氟-2-脱氧胞苷联合修饰；A2 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxycytidine;
A3为2-氟-2-脱氧尿苷和2-氟-2-脱氧鸟苷联合修饰；A3 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxyguanosine;
A4为2-氟-2-脱氧尿苷和5-甲基胞苷联合修饰；A4 is the combined modification of 2-fluoro-2-deoxyuridine and 5-methylcytidine;
A5为2-氟-2-脱氧尿苷和N7-甲基-鸟苷联合修饰；A5 is the combined modification of 2-fluoro-2-deoxyuridine and N7-methyl-guanosine;
A6为2-氟-2-脱氧尿苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；A6 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-5-methylcytidine;
A7为2-氟-2-脱氧尿苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；A7 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
A8为2-氟-2-脱氧尿苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；A8 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-N4-acetylcytidine;
A9为2-氟-2-脱氧尿苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；A9 is the combined modification of 2-fluoro-2-deoxyuridine and 2-fluoro-2-deoxy-N6-methyladenosine;
B1为假尿苷和2-氟-2-脱氧腺苷联合修饰；B1 is the combined modification of pseudouridine and 2-fluoro-2-deoxyadenosine;
B2为假尿苷和2-氟-2-脱氧胞苷联合修饰；B2 is the combined modification of pseudouridine and 2-fluoro-2-deoxycytidine;
B3为假尿苷和2-氟-2-脱氧鸟苷联合修饰；B3 is the combined modification of pseudouridine and 2-fluoro-2-deoxyguanosine;
B4为假尿苷和5-甲基胞苷联合修饰；B4 is the combined modification of pseudouridine and 5-methylcytidine;
B5为假尿苷和N7-甲基-鸟苷联合修饰；B5 is the combined modification of pseudouridine and N7-methyl-guanosine;
B6假尿苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；Combined modification of B6 pseudouridine and 2-fluoro-2-deoxy-5-methylcytidine;
B7假尿苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；Combined modification of B7 pseudouridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
B8假尿苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；Combined modification of B8 pseudouridine and 2-fluoro-2-deoxy-N4-acetylcytidine;
B9假尿苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；Combined modification of B9 pseudouridine and 2-fluoro-2-deoxy-N6-methyladenosine;
C1为N1-甲基-假尿苷和2-氟-2-脱氧腺苷联合修饰；C1 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxyadenosine;
C2为N1-甲基-假尿苷和2-氟-2-脱氧胞苷联合修饰；C2 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxycytidine;
C3为N1-甲基-假尿苷和2-氟-2-脱氧鸟苷联合修饰；C3 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxyguanosine;
C4为N1-甲基-假尿苷和5-甲基胞苷联合修饰；C4 is the combined modification of N1-methyl-pseudouridine and 5-methylcytidine;
C5为N1-甲基-假尿苷和N7-甲基-鸟苷联合修饰；C5 is the combined modification of N1-methyl-pseudouridine and N7-methyl-guanosine;
C6为N1-甲基-假尿苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；C6 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methylcytidine;
C7为N1-甲基-假尿苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；C7 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
C8为N1-甲基-假尿苷2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；C8 is the combined modification of N1-methyl-pseudouridine 2-fluoro-2-deoxy-N4-acetylcytidine;
C9为N1-甲基-假尿苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；C9 is the combined modification of N1-methyl-pseudouridine and 2-fluoro-2-deoxy-N6-methyladenosine;
D1为5-甲氧基尿苷和2-氟-2-脱氧腺苷联合修饰；D1 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxyadenosine;
D2为5-甲氧基尿苷和2-氟-2-脱氧胞苷联合修饰；D2 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxycytidine;
D3为5-甲氧基尿苷和2-氟-2-脱氧鸟苷联合修饰；D3 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxyguanosine;
D4为5-甲氧基尿苷和5-甲基胞苷联合修饰；D4 is the combined modification of 5-methoxyuridine and 5-methylcytidine;
D5为5-甲氧基尿苷和N7-甲基-鸟苷联合修饰；D5 is the combined modification of 5-methoxyuridine and N7-methyl-guanosine;
D6为5-甲氧基尿苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；D6 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-5-methylcytidine;
D7为5-甲氧基尿苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；D7 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
D8为5-甲氧基尿苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；D8 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-N4-acetylcytidine;
D9为5-甲氧基尿苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；D9 is the combined modification of 5-methoxyuridine and 2-fluoro-2-deoxy-N6-methyladenosine;
E1为N4-乙酰基胞苷和2-氟-2-脱氧腺苷联合修饰；E1 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxyadenosine;
E2为N4-乙酰基胞苷和2-氟-2-脱氧鸟苷联合修饰；E2 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxyguanosine;
E3为N4-乙酰基胞苷和N7-甲基-鸟苷联合修饰；E3 is the combined modification of N4-acetylcytidine and N7-methyl-guanosine;
E4为N4-乙酰基胞苷和2-氟-2-脱氧-假尿苷联合修饰；E4 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-pseudouridine;
E5为N4-乙酰基胞苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；E5 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
E6为N4-乙酰基胞苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；E6 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
E7为N4-乙酰基胞苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；E7 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-5-methoxyuridine;
E8为N4-乙酰基胞苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；E8 is the combined modification of N4-acetylcytidine and 2-fluoro-2-deoxy-N6-methyladenosine;
F1为N6-甲基腺苷和2-氟-2-脱氧胞苷联合修饰；F1 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxycytidine;
F2为N6-甲基腺苷和2-氟-2-脱氧鸟苷联合修饰；F2 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxyguanosine;
F3为N6-甲基腺苷和5-甲基胞苷联合修饰；F3 is the combined modification of N6-methyladenosine and 5-methylcytidine;
F4为N6-甲基腺苷和N7-甲基-鸟苷联合修饰；F4 is the combined modification of N6-methyladenosine and N7-methyl-guanosine;
F5为N6-甲基腺苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；F5 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-5-methylcytidine;
F6为N6-甲基腺苷和2-氟-2-脱氧-假尿苷联合修饰；F6 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-pseudouridine;
F7为N6-甲基腺苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；F7 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
F8为N6-甲基腺苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；F8 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
F9为N6-甲基腺苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；F9 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-5-methoxyuridine;
F10为N6-甲基腺苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；F10 is the combined modification of N6-methyladenosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
G1为2-氟-2-脱氧胞苷和2-氟-2-脱氧-假尿苷联合修饰；G1 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-pseudouridine;
G2为2-氟-2-脱氧胞苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；G2 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
G3为2-氟-2-脱氧胞苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；G3 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
G4为2-氟-2-脱氧胞苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；G4 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-5-methoxyuridine;
G5为2-氟-2-脱氧胞苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；G5 is the combined modification of 2-fluoro-2-deoxycytidine and 2-fluoro-2-deoxy-N6-methyladenosine;
G6为2-氟-2-脱氧鸟苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；G6 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-5-methylcytidine;
G7为2-氟-2-脱氧鸟苷和2-氟-2-脱氧-假尿苷联合修饰；G7 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-pseudouridine;
G8为2-氟-2-脱氧鸟苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；G8 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
G9为2-氟-2-脱氧鸟苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；G9 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-5-methoxyuridine;
G10为2-氟-2-脱氧鸟苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；G10 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
G11为2-氟-2-脱氧鸟苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；G11 is the combined modification of 2-fluoro-2-deoxyguanosine and 2-fluoro-2-deoxy-N6-methyladenosine;
H1为2-氟-2-脱氧腺苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；H1 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-5-methylcytidine;
H2为2-氟-2-脱氧腺苷和2-氟-2-脱氧-假尿苷联合修饰；H2 is a combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-pseudouridine;
H3为2-氟-2-脱氧腺苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；H3 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
H4为2-氟-2-脱氧腺苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；H4 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
H5为2-氟-2-脱氧腺苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；H5 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-5-methoxyuridine;
H6为2-氟-2-脱氧腺苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；H6 is the combined modification of 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
H7为5-甲基胞苷和2-氟-2-脱氧-假尿苷联合修饰；H7 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-pseudouridine;
H8为5-甲基胞苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；H8 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
H9为5-甲基胞苷和2-氟-2-脱氧-N7-甲基-鸟苷联合修饰；H9 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-N7-methyl-guanosine;
H10为5-甲基胞苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；H10 is the combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-5-methoxyuridine;
H11为5-甲基胞苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；H11 is a combined modification of 5-methylcytidine and 2-fluoro-2-deoxy-N6-methyladenosine;
H12为N7-甲基-鸟苷和2-氟-2-脱氧-5-甲基胞苷联合修饰；H12 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-5-methylcytidine;
H13为N7-甲基-鸟苷和2-氟-2-脱氧-假尿苷联合修饰；H13 is a combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-pseudouridine;
H14为N7-甲基-鸟苷和2-氟-2-脱氧-N1-甲基-假尿苷联合修饰；H14 is a combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-N1-methyl-pseudouridine;
H15为N7-甲基-鸟苷和2-氟-2-脱氧-5-甲氧基尿苷联合修饰；H15 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-5-methoxyuridine;
H16为N7-甲基-鸟苷和2-氟-2-脱氧-N4-乙酰基胞苷联合修饰；H16 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-N4-acetylcytidine;
H17为N7-甲基-鸟苷和2-氟-2-脱氧-N6-甲基腺苷联合修饰；H17 is the combined modification of N7-methyl-guanosine and 2-fluoro-2-deoxy-N6-methyladenosine;
加尾反应(可选)tailing reaction (optional)
若PCR法或线性质粒法中所得的DNA模板中不含有聚腺苷酸序列(polyA)，则此处需有加尾反应，具体步骤如下：If the DNA template obtained by PCR method or linear plasmid method does not contain polyadenylic acid sequence (polyA), a tailing reaction is required here. The specific steps are as follows:
1、室温下配制如下反应体系在50ml Tube管中；1. Prepare the following reaction system in a 50ml Tube at room temperature;
表4加尾反应体系Table 4 tailing reaction system
化学合成的mRNAchemically synthesized mRNA 20μl20μl
Nuclease-free waterNuclease-free water 36μl36μl
5×E-PAP Buffer5×E-PAP Buffer 20μl20μl
25nM MnCl 2 25nM MnCl2 10μl10μl
10nM ATP10nM ATP 10μl10μl
2、向所述加尾反应体系中加入4μl E-PAP酶，37℃孵育1h。2. Add 4 μl of E-PAP enzyme to the tailing reaction system, and incubate at 37°C for 1 h.
DNase I消化去除DNA模板DNase I digestion to remove DNA template
向mRNA的体外合成后的每个Tube管中各加入120μl DNase I。上下颠倒10次混匀，1000rpm离心10s。重新置于恒温反应器中，37℃，1h(恒温器热盖设置为42℃)。反应结束后，将反应液合并到RNase-free 50ml Tube管中，检测DNA片段的残留。Add 120 μl of DNase I to each Tube after in vitro synthesis of mRNA. Mix by inversion 10 times and centrifuge at 1000rpm for 10s. It was placed in a thermostatic reactor again at 37°C for 1 h (the thermostat hot lid was set to 42°C). After the reaction, the reaction solution was combined into an RNase-free 50ml Tube to detect the residue of DNA fragments.
mRNA沉淀回收mRNA pellet recovery
向加尾反应后的每个50ml Tube管中，加入等体积的醋酸铵溶液。上下颠倒10次混匀。置于-20℃ 2h，沉淀。17000g，4℃离心，30min。去掉上清，用70％乙醇洗涤沉淀。17000g，4℃离心，10min。去掉70％乙醇，于超净台中蒸干，每管加入RNase-free水20ml。静置10min后，用枪头轻吹混匀。To each 50ml Tube after the tailing reaction, add an equal volume of ammonium acetate solution. Mix by inverting 10 times. Placed at -20°C for 2h to precipitate. 17000g, 4°C centrifugation, 30min. Remove the supernatant and wash the pellet with 70% ethanol. Centrifuge at 17000g at 4°C for 10min. Remove 70% ethanol, evaporate to dryness in an ultra-clean bench, and add 20 ml of RNase-free water to each tube. After standing for 10 minutes, mix with a pipette tip.
用NanoDrop检测回收后的mRNA浓度，以及260/280、260/230的比值。The recovered mRNA concentration and the ratio of 260/280 and 260/230 were detected by NanoDrop.
取1μl，稀释10倍，进行RNA ScreenTape assay以及琼脂糖凝胶电泳检测其片段完整性。Take 1 μl, dilute it 10 times, and perform RNA ScreenTape assay and agarose gel electrophoresis to detect the integrity of the fragment.
FPLC纯化mRNAFPLC purified mRNA
将上一步骤中的mRNA进行FPLC纯化。The mRNA from the previous step was subjected to FPLC purification.
10mRNA超滤10mRNA ultrafiltration
Millipore 30Kd超滤FPLC纯化后的mRNA。Millipore 30Kd ultrafiltration FPLC-purified mRNA.
用生理盐水洗脱和溶解。Elute and dissolve with saline.
用NanoDrop检测溶解后的mRNA浓度，以及260/280、260/230的比值。The dissolved mRNA concentration and the ratio of 260/280 and 260/230 were detected by NanoDrop.
取1μl样，稀释10倍，进行RNA ScreenTape assay以及琼脂糖凝胶电泳检测其片段完整性。条带大小正确、清晰、无杂带、无降解视为片段完整。Take 1 μl of the sample, dilute it 10 times, and perform RNA ScreenTape assay and agarose gel electrophoresis to detect the integrity of the fragments. Bands with correct size, clearness, no stray bands, and no degradation were considered complete fragments.
实施例2Example 2
根据本发明的编码EPO的单一种类修饰或多种类修饰mRNA，检测其免疫原性，评价标准为修饰mRNA免疫沉淀测试(RIP分析)检测Toll样受体TRL3、TRL7、TRL8和RIG-1与mRNA的结合水平；具体步骤如下：According to the single-type modified or multiple-type modified mRNA encoding EPO of the present invention, its immunogenicity is detected, and the evaluation standard is the modified mRNA immunoprecipitation test (RIP analysis) to detect Toll-like receptors TRL3, TRL7, TRL8 and RIG-1 and mRNA The level of binding; the specific steps are as follows:
细胞转染cell transfection
接种完293T细胞(购自中国科学院细胞库)后约24h，观察6孔板内的细胞状态，汇合度在88％～92％。在生物安全柜内，配制90％(体积百分含量)DMEM+10％(体积百分含量)FBS培养基。转染前30min弃掉孔板的培养基，每孔加入1ml新鲜培养基，即90％(体积百分含量)DMEM+10％(体积百分含量)FBS培养基。About 24 hours after inoculation of 293T cells (purchased from the cell bank of the Chinese Academy of Sciences), the state of the cells in the 6-well plate was observed, and the confluence was 88%-92%. In a biological safety cabinet, prepare 90% (volume percent) DMEM+10% (volume percent) FBS medium. 30 min before transfection, the medium of the well plate was discarded, and 1 ml of fresh medium was added to each well, namely 90% (volume percentage) DMEM+10% (volume percentage) FBS medium.
配制转染体系：取200μl opti-MEM，加入10μg供试品(浓度2μg/μl，5μl)或阴性对照GFP-mRNA，用枪头轻轻吹打混匀，再加入60μl PEI(浓度1mg/ml)，立即置于漩涡振荡器上振荡10次，每次1s，充分混匀，静置10min。将配制好的转染体系，直接均匀滴加进入培养的细胞中，再前后左右摇匀，使得转染体系均匀分布于细胞上。转染后6h换液，吸掉旧的培养基，每孔换为2ml新鲜培养基(90％DMEM+10％FBS)。转染后30～36h收获。吸掉旧的培养基，用1ml PBS清洗一遍。吸掉PBS，继续用1ml PBS将细胞吹打下来，收集于1.5ml离心管中，300g离心5min。将离心后的上清尽量吸去干净，沉淀的细胞用于检测。Preparation of transfection system: take 200μl opti-MEM, add 10μg test substance (concentration 2μg/μl, 5μl) or negative control GFP-mRNA, mix by pipetting gently, then add 60μl PEI (concentration 1mg/ml) , immediately placed on a vortex shaker for 10 times, 1 s each time, mixed well, and let stand for 10 min. The prepared transfection system is directly and evenly dropped into the cultured cells, and then shaken up and down, so that the transfection system is evenly distributed on the cells. 6h after transfection, the medium was changed, the old medium was aspirated, and each well was replaced with 2 ml of fresh medium (90% DMEM+10% FBS). Harvest 30-36 h after transfection. Aspirate the old medium and wash with 1ml PBS. Aspirate the PBS, continue to pipet down the cells with 1ml PBS, collect them in a 1.5ml centrifuge tube, and centrifuge at 300g for 5min. The supernatant after centrifugation was aspirated as cleanly as possible, and the precipitated cells were used for detection.
细胞因子检测Cytokine detection
用转染各种mRNA后获得的细胞，研究人PBMCs中TNFα和IL-8水平。TNFα and IL-8 levels in human PBMCs were studied using cells obtained after transfection of various mRNAs.
用人类IL-8和TNFa试剂盒(RayBio)进行酶联免疫吸附分析(elisa)。Enzyme-linked immunosorbent assay (elisa) was performed with human IL-8 and TNFa kit (RayBio).
RNA免疫沉淀RNA immunoprecipitation
1、用10μg mRNA转染10 6个人PBMCs细胞，24h之后消化细胞，400rpm 10min离心沉淀细胞。用与细胞等体积的RIP裂解液重悬细胞，吹打均匀后于冰上静置5min。每管分装200μl细胞裂解液，贮存于-80℃ 1. Transfect 10 6 human PBMCs cells with 10 μg mRNA, digest the cells after 24 h, and centrifuge the cells at 400 rpm for 10 min to pellet the cells. Cells were resuspended with an equal volume of RIP lysis buffer, pipetted evenly, and then placed on ice for 5 min. Aliquot 200 μl of cell lysate per tube and store at -80°C
2、吸取50μl重悬后的磁珠悬液于每个enpendoff管，每管加入500μl RIP Wash Buffer，涡旋震荡，将enpendoff管 置于磁力架上，并左右转动15°使磁珠吸附成一条直线，去上清，重复一次。用100μl的RIP Wash Buffer重悬磁珠，分别加入约5μg TRL3、TRL7、TRL8和RIG-1于每个样品中，室温孵育30min。将enpendoff管置于磁力架上，弃上清。加入500μl RIP Wash Buffer，涡旋震荡后置于冰上。2. Pipet 50μl of the resuspended magnetic bead suspension into each enpendoff tube, add 500μl RIP Wash Buffer to each tube, vortex and shake, place the enpendoff tube on the magnetic stand, and turn 15° left and right to make the magnetic beads adsorb into a line Straight line, go to supernatant, repeat once. Resuspend the magnetic beads with 100 μl of RIP Wash Buffer, add about 5 μg of TRL3, TRL7, TRL8 and RIG-1 to each sample, and incubate at room temperature for 30 min. Place the enpendoff tube on the magnetic stand and discard the supernatant. Add 500 μl RIP Wash Buffer, vortex and place on ice.
3、将前上步的enpendoff管放磁力架上，去上清，每管加入900μl RIP Immunoprecipitation Buffer迅速解冻第一步制备的细胞裂解液，14,000rpm，4℃离心10min。吸取100μl上清液于上一步的磁珠-抗体复合物中，使得总体积为1ml。4℃孵育过夜。短暂离心，将enpendoff管放在磁力架上，弃上清。加入500μl RIP Wash Buffer，涡旋震荡后将enpendoff管放在磁力架上，弃上清，重复清洗6次。3. Put the enpendoff tube in the previous step on the magnetic stand, remove the supernatant, add 900μl RIP Immunoprecipitation Buffer to each tube and quickly thaw the cell lysate prepared in the first step, centrifuge at 14,000rpm and 4°C for 10min. Pipette 100 μl of the supernatant into the magnetic bead-antibody complex from the previous step to make a total volume of 1 ml. Incubate overnight at 4°C. Briefly centrifuge, place the enpendoff tube on the magnetic stand, and discard the supernatant. Add 500μl RIP Wash Buffer, vortex and place the enpendoff tube on the magnetic stand, discard the supernatant, and repeat the washing 6 times.
4、用150μl Proteinase K Buffer重悬上述磁珠-抗体复合物55℃孵育30min。孵育完之后，将enpendoff管置于磁力架上，将上清液吸入一新的enpendoff管中。于每管上清液中加入250μl RIP Wash Buffer。于每管加入400μl苯酚：氯仿：异戊醇，涡旋震荡15s，室温下14,000rpm离心10min。小心的吸取350μl上层水相，吸入另一新的enpendoff管。于每管加入400μl氯仿，涡旋震荡15s，室温下14,000rpm离心10min。小心的吸取300μl上层水相，吸入另一新的enpendoff管。每管加入50μl Salt SolutionⅠ，15μl Salt SolutionⅡ，5μl Precipitate Enhancer，850μl无水乙醇(无RNase)，混合，-80℃保持过夜。14,000rpm，4℃离心30min，小心去上清。用80％乙醇冲洗一次，14,000rpm，4℃离心15min，小心去上清，空气中晾干。10～20μl DEPC水溶解，-80℃保存或进行下游qPCR实验。4. Resuspend the above magnetic bead-antibody complex with 150μl Proteinase K Buffer and incubate at 55°C for 30min. After incubation, place the enpendoff tube on the magnetic stand and aspirate the supernatant into a new enpendoff tube. Add 250 μl RIP Wash Buffer to each tube of supernatant. Add 400 μl of phenol:chloroform:isoamyl alcohol to each tube, vortex for 15s, and centrifuge at 14,000rpm for 10min at room temperature. Carefully pipette 350 μl of the upper aqueous phase into another new enpendoff tube. Add 400 μl of chloroform to each tube, vortex for 15 s, and centrifuge at 14,000 rpm for 10 min at room temperature. Carefully pipette 300 μl of the upper aqueous phase into another new enpendoff tube. Add 50 μl Salt Solution I, 15 μl Salt Solution II, 5 μl Precipitate Enhancer, 850 μl absolute ethanol (RNase free) to each tube, mix, and keep at -80°C overnight. Centrifuge at 14,000 rpm for 30 min at 4°C and carefully remove the supernatant. Rinse once with 80% ethanol, centrifuge at 14,000 rpm for 15 min at 4°C, carefully remove the supernatant, and air dry. Dissolve in 10-20μl DEPC water, store at -80°C or perform downstream qPCR experiments.
定量RT-PCRqRT-PCR
将RIP所得的RNA样品利用takara逆转录试剂盒进行cDNA合成，利用BioRad SYBR GREEN kit进行定量PCR实验，计算不修饰mRNA、单一种类修饰mRNA和多种类修饰mRNA样品之间的拷贝数关系。The RNA samples obtained by RIP were used for cDNA synthesis using the takara reverse transcription kit, and quantitative PCR experiments were performed using the BioRad SYBR GREEN kit to calculate the copy number relationship between unmodified mRNA, single type of modified mRNA and multiple types of modified mRNA samples.
结果如图1～37所示，本发明所述的化学修饰EPO mRNA与TRL3、TRL7、TRL8和RIG-1的结合相对于未修饰的EPO mRNA显著减少，且TNFα和IL-8水平也显著减少，多种类修饰比单种类修饰明显更有效。本实施例表明基于本发明的单一种类修饰和多种类修饰的mRNA显著减少Toll样受体的结合，从而减少免疫应答，使这些修饰mRNA能够更好的用于体内诊断或治疗。The results are shown in Figures 1 to 37, the binding of chemically modified EPO mRNA to TRL3, TRL7, TRL8 and RIG-1 was significantly reduced compared to unmodified EPO mRNA, and the levels of TNFα and IL-8 were also significantly reduced. , multi-species modification is significantly more effective than single-species modification. This example shows that single-type modified and multiple-type modified mRNAs based on the present invention significantly reduce the binding of Toll-like receptors, thereby reducing immune responses, so that these modified mRNAs can be better used for in vivo diagnosis or treatment.
实施例3Example 3
根据本发明的编码EPO的单修饰或多修饰mRNA，检测其免疫原性，评价标准为修饰mRNA对小鼠进行肌肉注射后血清中TNFα和IL-8水平。According to the single-modified or multi-modified mRNA encoding EPO of the present invention, its immunogenicity is detected, and the evaluation standard is the level of TNFα and IL-8 in serum after intramuscular injection of the modified mRNA into mice.
将6-8周龄的balbc小鼠(购自北京维通利华实验动物技术有限公司)在SPF条件下，并且保持12h光亮和12h黑暗循环下的通气笼中饲养，将以上单一种类修饰或多种类修饰的EPO mRNA对balb/c小鼠进行肌肉注射，每只小鼠的注射剂量为100μg，24h后对小鼠进行眼眶取血，分离血清。用小鼠IL-8和TNFa试剂盒(RayBio)进行酶联免疫吸附分析(elisa)。6-8-week-old balbc mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were kept under SPF conditions and kept in ventilated cages under 12h light and 12h dark cycles. Various types of modified EPO mRNA were injected intramuscularly into balb/c mice, and the injection dose of each mouse was 100 μg. After 24 hours, the orbital blood was collected from the mice, and the serum was separated. Enzyme-linked immunosorbent assay (elisa) was performed with mouse IL-8 and TNFa kit (RayBio).
结果如图46～63所示，在小鼠体内，本发明提供的修饰策略合成的mRNA的免疫原性远远小于对照组未修饰的核酸。The results are shown in Figures 46-63, in mice, the immunogenicity of the mRNA synthesized by the modification strategy provided by the present invention is far less than that of the unmodified nucleic acid in the control group.
实施例4Example 4
将上述实施例中制备的修饰荧光素酶(Luc)mRNA，通过气管内给药的高压喷雾装置直接引入小鼠肺部，体内生物荧光信号表征修饰mRNA在体内的表达强度和表达时间。The modified luciferase (Luc) mRNA prepared in the above example was directly introduced into the lungs of mice through a high-pressure spray device for intratracheal administration, and the in vivo bioluminescence signal characterized the expression intensity and expression time of the modified mRNA in vivo.
雾化器气管内给药Nebulizer for intratracheal drug delivery
将Balb/c小鼠戊巴比妥钠麻醉，腹部朝上固定在注射平台上，使得上齿的角度为45°，利用小型压舌板打开小鼠下颌，并用钝头夹钳将舌头牵引至一侧从而暴露口咽部，将高压雾化器针头插入气管，连续施用30μl Luc mRNA(1μg/μl)溶液，随后取走小鼠。The Balb/c mice were anesthetized with sodium pentobarbital, and the abdomen was fixed on the injection platform so that the angle of the upper teeth was 45°. A small tongue depressor was used to open the lower jaw of the mouse, and the tongue was pulled with blunt forceps. With one side exposed to expose the oropharynx, a high pressure nebulizer needle was inserted into the trachea, 30 μl of Luc mRNA (1 μg/μl) solution was administered continuously, and the mice were removed.
小动物成像Small Animal Imaging
将D-荧光素底物溶解于生理盐水，浓度为15mg/ml，将100μl该溶液经尾静脉注射进入小鼠体内。10min后，使用IVIS小动物成像系统定量分析肺部信号强弱。The D-luciferin substrate was dissolved in physiological saline at a concentration of 15 mg/ml, and 100 μl of this solution was injected into mice via tail vein. After 10 min, the signal intensity in the lungs was quantitatively analyzed using the IVIS small animal imaging system.
结果如下图55～63所示，3h即可检测到体内荧光素酶表达，24h不修饰Luc mRNA组荧光开始下降，3天降到无法检测水平，而单修饰和多修饰分别持续观察到高表达值直到10天。The results are shown in Figures 55-63 below. The expression of luciferase in vivo can be detected within 3 hours. The fluorescence of the unmodified Luc mRNA group began to decline after 24 hours, and dropped to an undetectable level within 3 days, while the single-modification and multi-modification continued to observe high expression respectively. Value until 10 days.
实施例5Example 5
通过检测血液中EPO蛋白含量及红细胞压积值来评估上述实施例中制备的化学修饰的EPO的mRNA的表达效力。The expression efficiency of the mRNA of chemically modified EPO prepared in the above examples was evaluated by detecting the EPO protein content and hematocrit value in the blood.
100μg未修饰和100μg修饰EPO mRNA经尾静脉注射进入h体内，24h后对小鼠进行眼眶取血，进行ELISA实验检测EPO含量，以及7d检测小鼠全血的红细胞压积值。100μg unmodified and 100μg modified EPO mRNA were injected into the body via tail vein injection. After 24 hours, the orbital blood was collected from the mice to detect the EPO content by ELISA and the hematocrit value of the whole blood of mice at 7 days.
ELISA检测EPO含量ELISA detection of EPO content
1.标准品稀释：用coating buffer将EPO标准品依次稀释到ST1：3857ng/ml、ST2：1928ng/ml、ST3：964ng/ml、 ST4：482ng/ml、ST5：241ng/ml、ST6：121ng/ml，以coating buffer为阴性对照。1. Standard dilution: Dilute the EPO standard with coating buffer to ST1: 3857ng/ml, ST2: 1928ng/ml, ST3: 964ng/ml, ST4: 482ng/ml, ST5: 241ng/ml, ST6: 121ng/ml ml, with coating buffer as negative control.
2.包被：取96孔板依次加入标准品溶液、小鼠血清、阴性对照100μl/孔，平行2孔。2～8℃封闭过夜。2. Coating: Take a 96-well plate and add 100 μl/well of standard solution, mouse serum, and negative control in sequence, parallel to 2 wells. Block overnight at 2-8°C.
3.洗涤液(1×)配制：用灭菌注射用水将50×的Washing buffer稀释50倍，混匀，即得。3. Preparation of washing solution (1×): Dilute 50× Washing buffer 50 times with sterile water for injection, and mix well.
4.洗板：从4℃冰箱取出包被好的96孔板，将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。4. Wash the plate: Take out the coated 96-well plate from the refrigerator at 4°C, pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add 300 μl of washing solution (1×) to each well, and let it stand for a while. Set for 30s, pour the liquid in the wells into the waste liquid tank, and repeat the plate washing 4 times to pat the wells dry.
5.封闭：加入Blocking buffer，250μl/孔，封上封板膜，室温封闭2h。5. Blocking: Add Blocking buffer, 250μl/well, seal with sealing film, and block at room temperature for 2h.
6.洗板：将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入约300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。6. Wash the plate: pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 μl of washing solution (1×) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
7.用dilution buffer将EPO Rab(abcam)稀释1000倍，100μl/孔，封上封板膜，室温孵育1.5h。7. Dilute EPO Rab (abcam) by 1000 times with dilution buffer, 100 μl/well, seal with sealing film, and incubate at room temperature for 1.5 h.
8.洗板：将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入约300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。8. Wash the plate: pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 μl of washing solution (1×) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
9.用dilution buffer将GoatpAb to Rb IgG(HRP)稀释10000倍，100μl/孔，封上封板膜，室温孵育1h。9. Dilute GoatpAb to Rb IgG(HRP) 10000 times with dilution buffer, 100μl/well, seal with sealing film, and incubate at room temperature for 1h.
10.洗板：将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入约300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。10. Wash the plate: pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 μl of washing solution (1×) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
11.加TMB buffer，100μl/孔。混匀，置室温避光孵育20min。11. Add TMB buffer, 100μl/well. Mix well and incubate at room temperature for 20 min in the dark.
12.孵育结束后，每孔加入Stop buffer 100μl。12. After the incubation, add 100 μl of Stop buffer to each well.
13.尽快将孔板放入酶标仪，于450nn波长下测定吸光度。13. Put the plate into the microplate reader as soon as possible, and measure the absorbance at the wavelength of 450nm.
14.数据处理：以标准品的平均OD值为纵坐标，浓度为横坐标，采用四参数逻辑拟合方程。14. Data processing: Take the average OD value of the standard product as the ordinate and the concentration as the abscissa, and use a four-parameter logistic fitting equation.
红细胞压积测定Hematocrit determination
取小鼠血液300μl，加入10μl肝素钠抗凝，混匀后加入温氏血沉管，3100g离心30min，记录红细胞所占体积比例。Take 300 μl of mouse blood, add 10 μl of heparin sodium for anticoagulation, mix well, add to Wen's ESR, centrifuge at 3100 g for 30 min, and record the volume ratio of red blood cells.
结果如图64～81所示，不修饰的EPO mRNA表达量不高，而修饰的EPO mRNA非常稳定，除了持续表达产生EPO蛋白之外，红细胞压积持续升高，可被用于治疗促红细胞生成缺陷。多种类修饰mRNA稳定性和表达效力高于单种类修饰mRNA。The results are shown in Figures 64 to 81. The expression of unmodified EPO mRNA is not high, while the modified EPO mRNA is very stable. In addition to the continuous expression of EPO protein, the hematocrit continues to increase, which can be used to treat erythropoietic cells. Generate defects. The stability and expression efficiency of multiple types of modified mRNAs were higher than those of single type of modified mRNAs.
实施例6Example 6
将制备的EPO mRNA溶解在RNase-free水、生理盐水、柠檬酸缓冲液以及柠檬酸-生理盐水缓冲液中，初始浓度为2224ng/μl。包装在1ml中性硼硅西林瓶、注射液用卤化丁基胶塞11-B以及铝盖轧盖。考察mRNA在2～8℃\强光(4500±500Lx)条件下，其主要成分mRNA浓度(含量)、纯度、pH的稳定性。The prepared EPO mRNA was dissolved in RNase-free water, physiological saline, citrate buffer and citrate-physiological saline buffer at an initial concentration of 2224ng/μl. Packaged in a 1ml neutral borosilicate bottle, a halogenated butyl rubber stopper 11-B for injection, and an aluminum cap crimp. Investigate the stability of mRNA concentration (content), purity and pH of its main components under the conditions of 2～8℃\strong light (4500±500Lx).
考察结果如图82～84所示，不同赋形剂中的mRNA在2～8℃，4500±500Lx条件下放置5个月条件，纯度和浓度有下降趋势。The investigation results are shown in Figures 82-84. The mRNA in different excipients was placed under the conditions of 2-8°C and 4500±500Lx for 5 months, and the purity and concentration showed a downward trend.
溶解在水中的mRNA纯度由原来的99.3％下降至93.2％，整体下降了6.14％，浓度由原来的2224ng/μl下降至1900ng/μl，整体下降了14.57％，pH值上升0.9。The purity of mRNA dissolved in water decreased from 99.3% to 93.2%, the overall decrease was 6.14%, the concentration decreased from 2224ng/μl to 1900ng/μl, the overall decrease was 14.57%, and the pH value increased by 0.9.
溶解在生理盐水中的mRNA纯度由原来的99.2％下降至94.0％，整体下降了5.24％，浓度由原来的2224ng/μl下降至1924ng/μl，整体下降了13.49％，pH值上升0.7。The purity of mRNA dissolved in normal saline decreased from 99.2% to 94.0%, the overall decrease was 5.24%, the concentration decreased from 2224ng/μl to 1924ng/μl, the overall decrease was 13.49%, and the pH value increased by 0.7.
溶解在柠檬酸缓冲液中的mRNA纯度由原来的99.3％下降至95.1％，整体下降了4.23％，浓度由原来的2224ng/μl下降至1947ng/μl，整体下降了12.46％，pH值上升0.8。The purity of mRNA dissolved in citrate buffer decreased from 99.3% to 95.1%, the overall decrease was 4.23%, the concentration decreased from 2224ng/μl to 1947ng/μl, the overall decrease was 12.46%, and the pH value increased by 0.8.
溶解在柠檬酸生理盐水缓冲液中的mRNA纯度由原来的99.3％下降至96.7％，整体下降了2.62％，浓度由原来的2224ng/μl下降至2048ng/μl，整体下降了7.91％，pH值上升0.3。The purity of mRNA dissolved in citrate saline buffer decreased from 99.3% to 96.7%, the overall decrease was 2.62%, the concentration decreased from 2224ng/μl to 2048ng/μl, the overall decrease was 7.91%, and the pH value increased 0.3.
实施例7Example 7
在合成Luc mRNA时，设置以下实验组，分别为无修饰组、25％假尿嘧啶+75％尿嘧啶、50％假尿嘧啶+50％尿嘧啶、75％假尿嘧啶+25％尿嘧啶、完全假尿嘧啶替代组。When synthesizing Luc mRNA, the following experimental groups were set, namely, no modification group, 25% pseudouracil+75% uracil, 50% pseudouracil+50% uracil, 75% pseudouracil+25% uracil, Complete pseudouracil replacement group.
将上述实施例中制备的修饰荧光素酶(Luc)mRNA，通过气管内给药的高压喷雾装置直接引入小鼠肺部，体内生物荧光信号表征修饰mRNA在体内的表达强度和表达时间。The modified luciferase (Luc) mRNA prepared in the above example was directly introduced into the lungs of mice through a high-pressure spray device for intratracheal administration, and the in vivo bioluminescence signal characterized the expression intensity and expression time of the modified mRNA in vivo.
雾化器气管内给药Nebulizer for intratracheal drug delivery
将Balb/c小鼠(购自北京维通利华实验动物技术有限公司)戊巴比妥钠麻醉，腹部朝上固定在注射平台上，使得上齿的角度为45°，利用小型压舌板打开小鼠下颌，并用钝头夹钳将舌头牵引至一侧从而暴露口咽部，将高压雾化器针头插入气管，连续施用30μl Luc mRNA(1μg/μl)溶液，随后取走小鼠。24h后检测荧光素酶在小鼠体内的表达情况。Balb/c mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were anesthetized with sodium pentobarbital, and fixed on the injection platform with the abdomen facing upward, so that the angle of the upper teeth was 45°, and a small tongue depressor was used. The mouse jaws were opened and the oropharynx was exposed by pulling the tongue to one side with blunt clamps, a high pressure nebulizer needle was inserted into the trachea, 30 μl of Luc mRNA (1 μg/μl) solution was administered continuously, and the mice were removed. The expression of luciferase in mice was detected after 24h.
小动物成像Small Animal Imaging
将D-荧光素底物溶解于生理盐水，浓度为15mg/ml，将100μl该溶液经尾静脉注射进入小鼠体内。10min后， 使用IVIS小动物成像系统定量分析肺部信号强弱。The D-luciferin substrate was dissolved in physiological saline at a concentration of 15 mg/ml, and 100 μl of this solution was injected into mice via tail vein. After 10 min, use the IVIS small animal imaging system to quantitatively analyze the signal intensity in the lungs.
结果如图84所示，修饰碱基的掺入比例与mRNA表达水平的高低具有显著相关性，修饰碱基的掺入比例越高，mRNA的表达水平越高。The results are shown in FIG. 84 , the incorporation ratio of modified bases was significantly correlated with the level of mRNA expression, and the higher the incorporation ratio of modified bases, the higher the mRNA expression level.
实施例8Example 8
人工合成编码新型冠状病毒2019-nCov的刺突蛋白(S)的mRNA(核苷酸序列如SEQ ID No.11所示)，修饰方案为不修饰、2-氟-2-脱氧尿苷/N4-乙酰基胞苷/N7-甲基鸟苷/N6-甲基腺苷单独分别修饰以及四种碱基完全修饰。通过检测血液中S蛋白特异性抗体滴度来评估本实施例中制备的化学修饰的mRNA的表达效力差异。Artificially synthesized mRNA encoding the spike protein (S) of the novel coronavirus 2019-nCov (the nucleotide sequence is shown in SEQ ID No. 11), and the modification scheme is unmodified, 2-fluoro-2-deoxyuridine/N4 -Acetylcytidine/N7-methylguanosine/N6-methyladenosine are individually modified and four bases are fully modified. The difference in expression potency of the chemically modified mRNA prepared in this example was evaluated by detecting the S protein-specific antibody titer in blood.
100μg未修饰和100μg修饰S蛋白的mRNA经肌肉注射进入小鼠体内，4周后对小鼠进行眼眶取血，进行ELISA实验检测抗体滴度。100μg unmodified and 100μg modified S protein mRNA were injected into mice by intramuscular injection. After 4 weeks, orbital blood was collected from mice, and ELISA experiments were performed to detect antibody titers.
ELISA检测抗体滴度ELISA to detect antibody titers
1.标准品稀释：用coating buffer将S蛋白标准品稀释200ng/ml以coating buffer为阴性对照。1. Standard dilution: Dilute the S protein standard by 200ng/ml with coating buffer and use the coating buffer as a negative control.
2.包被：取96孔板依次加入标准品溶液、阴性对照100μl/孔，平行2孔。2～8℃封闭过夜。2. Coating: Take a 96-well plate and add 100 μl/well of standard solution and negative control in sequence, parallel to 2 wells. Block overnight at 2-8°C.
3.洗涤液(1×)配制：用灭菌注射用水将50×的Washing buffer稀释50倍，混匀，即得。3. Preparation of washing solution (1×): Dilute 50× Washing buffer 50 times with sterile water for injection, and mix well.
4.洗板：从4℃冰箱取出包被好的96孔板，将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。4. Wash the plate: Take out the coated 96-well plate from the refrigerator at 4°C, pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add 300 μl of washing solution (1×) to each well, and let it stand for a while. Set for 30s, pour the liquid in the wells into the waste liquid tank, and repeat the plate washing 4 times to pat the wells dry.
5.封闭：加入Blocking buffer，250μl/孔，封上封板膜，室温封闭2h。5. Blocking: Add Blocking buffer, 250μl/well, seal with sealing film, and block at room temperature for 2h.
6.洗板：将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入约300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。6. Wash the plate: pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 μl of washing solution (1×) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
7.用dilution buffer将小鼠血清稀释40、400、4000、40000、400000倍，100μl/孔，封上封板膜，室温孵育1.5h。7. Dilute the mouse serum 40, 400, 4000, 40000, 400000 times with dilution buffer, 100 μl/well, seal with a sealing film, and incubate at room temperature for 1.5 h.
8.洗板：将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入约300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。8. Wash the plate: pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 μl of washing solution (1×) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
9.用dilution buffer将Goat pAb to mouse IgG(HRP)稀释10000倍，100μl/孔，封上封板膜，室温孵育1h。9. Dilute Goat pAb to mouse IgG(HRP) 10000 times with dilution buffer, 100μl/well, seal with sealing film, and incubate at room temperature for 1h.
10.洗板：将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入约300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。10. Wash the plate: pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 μl of washing solution (1×) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
11.加TMB buffer，100μl/孔。混匀，置室温避光孵育20min。11. Add TMB buffer, 100μl/well. Mix well and incubate at room temperature for 20 min in the dark.
12.孵育结束后，每孔加入Stop buffer 100μl。12. After the incubation, add 100 μl of Stop buffer to each well.
13.尽快将孔板放入酶标仪，于450nn波长下测定吸光度。13. Put the plate into the microplate reader as soon as possible, and measure the absorbance at the wavelength of 450nm.
14.数据处理：以标准品的平均OD值为纵坐标，浓度为横坐标，采用四参数逻辑拟合方程。14. Data processing: Take the average OD value of the standard product as the ordinate and the concentration as the abscissa, and use a four-parameter logistic fitting equation.
实验结果如图85所示，mRNA部分碱基修饰和全碱基修饰时，表达效率具有较大差异，全碱基修饰的表达效率高于部分碱基修饰的表达效率。The experimental results are shown in Figure 85. When the mRNA is partially modified with bases and modified with all bases, the expression efficiency is quite different, and the expression efficiency of all base modifications is higher than that of partial base modifications.
实施例9Example 9
人工合成编码新型冠状病毒2019-nCov的刺突蛋白受体结合区(RBD)的mRNA(核苷酸序列如SEQ ID No.12所示)，修饰方案为不修饰、2-氟-2-脱氧尿苷/N4-乙酰基胞苷/2-氟-2-脱氧鸟苷/N6-甲基腺苷单独分别修饰以及四种碱基完全修饰。通过检测血液中RBD蛋白浓度以及特异性抗体滴度来评估本实施例中制备的化学修饰的mRNA的表达效力差异。Artificially synthesized mRNA encoding the spike protein receptor binding region (RBD) of the novel coronavirus 2019-nCov (the nucleotide sequence is shown in SEQ ID No. 12), and the modification scheme is unmodified, 2-fluoro-2-deoxy Uridine/N4-acetylcytidine/2-fluoro-2-deoxyguanosine/N6-methyladenosine were individually modified and four bases were fully modified. The difference in expression potency of the chemically modified mRNA prepared in this example was evaluated by detecting the RBD protein concentration and specific antibody titer in blood.
100μg未修饰和100μg修饰RBD蛋白的mRNA经肌肉注射进入小鼠体内，24h后对小鼠进行眼眶取血，进行ELISA实验检测RBD浓度。同时2周后再次眼眶取血，检测RBD抗体滴度。The mRNA of 100μg unmodified and 100μg modified RBD protein was injected into mice by intramuscular injection. After 24 hours, the orbital blood was collected from the mice, and the concentration of RBD was detected by ELISA experiment. At the same time, the orbital blood was collected again 2 weeks later to detect the RBD antibody titer.
ELISA检测RBD浓度：ELISA to detect RBD concentration:
1.标准品稀释：用coating buffer将RBD蛋白标准品稀释成400ng/ml，并依次10倍稀释6个梯度，以coating buffer为阴性对照。1. Standard dilution: use coating buffer to dilute the RBD protein standard to 400ng/ml, and dilute 6 gradients 10 times in sequence, with coating buffer as negative control.
2.包被：取96孔板依次加入7个浓度梯度的标准品溶液、小鼠血清、阴性对照，100μl/孔，平行2孔。2～8℃封闭过夜。2. Coating: Take a 96-well plate and add 7 concentration gradients of standard solution, mouse serum, and negative control in sequence, 100 μl/well, and 2 wells in parallel. Block overnight at 2-8°C.
3.洗涤液(1×)配制：用灭菌注射用水将50×的Washing buffer稀释50倍，混匀，即得。3. Preparation of washing solution (1×): Dilute 50× Washing buffer 50 times with sterile water for injection, and mix well.
4.洗板：从4℃冰箱取出包被好的96孔板，将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。4. Wash the plate: Take out the coated 96-well plate from the refrigerator at 4°C, pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add 300 μl of washing solution (1×) to each well, and let it stand for a while. Set for 30s, pour the liquid in the wells into the waste liquid tank, and repeat the plate washing 4 times to pat the wells dry.
5.封闭：加入Blocking buffer，250μl/孔，封上封板膜，室温封闭2h。5. Blocking: Add Blocking buffer, 250μl/well, seal with sealing film, and block at room temperature for 2h.
6.洗板：将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。6. Wash the plate: pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add 300 μl of washing solution (1×) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid tank , repeat the plate washing 4 times and pat the wells dry.
7.用dilution buffer将兔抗RBD一抗稀释1000倍，加入200μl，封上封板膜，室温孵育1.5h。7. Dilute rabbit anti-RBD primary antibody by 1000 times with dilution buffer, add 200 μl, seal with sealing film, and incubate at room temperature for 1.5 h.
8.洗板：将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入约300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。8. Wash the plate: pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 μl of washing solution (1×) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
9.用dilution buffer将Goat pAb to rabbitIgG(HRP)稀释10000倍，200μl/孔，封上封板膜，室温孵育1h。9. Dilute Goat pAb to rabbitIgG(HRP) 10000 times with dilution buffer, 200μl/well, seal with sealing film, and incubate at room temperature for 1h.
10.洗板：将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入约300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。10. Wash the plate: pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 μl of washing solution (1×) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
11.加TMB buffer，100μl/孔。混匀，置室温避光孵育20min。11. Add TMB buffer, 100μl/well. Mix well and incubate at room temperature for 20 min in the dark.
12.孵育结束后，每孔加入Stopbuffer 100μl。12. After the incubation, add 100 μl of Stopbuffer to each well.
13.尽快将孔板放入酶标仪，于450nn波长下测定吸光度。13. Put the plate into the microplate reader as soon as possible, and measure the absorbance at the wavelength of 450nm.
14.数据处理：以标准品的平均OD值为纵坐标，浓度为横坐标，采用四参数逻辑拟合方程。14. Data processing: Take the average OD value of the standard product as the ordinate and the concentration as the abscissa, and use a four-parameter logistic fitting equation.
Elisa检测RBD抗体滴度Elisa detects RBD antibody titers
除标准品为RBD蛋白外，与实施例8测定S蛋白抗体滴度方法相同。The method for determining the antibody titer of S protein in Example 8 is the same except that the standard substance is RBD protein.
实验结果如图86～87所示，本发明所示的化学修饰碱基能够显著增加RBD mRNA在小鼠体内的表达强度，从而引发更高的免疫反应。The experimental results are shown in Figures 86-87, the chemically modified bases shown in the present invention can significantly increase the expression intensity of RBD mRNA in mice, thereby triggering a higher immune response.
实施例10Example 10
人工合成编码人转化生长因子TGFβ3的mRNA(核苷酸序列如SEQ ID No.13所示)，修饰方案为不修饰及本发明中所有碱基单独修饰。通过检测细胞中TGFβ3蛋白的含量评估本实施例中制备的化学修饰的mRNA的表达效力差异。Artificially synthesized mRNA encoding human transforming growth factor TGFβ3 (the nucleotide sequence is shown in SEQ ID No. 13), and the modification scheme is no modification and all bases in the present invention are individually modified. The difference in expression potency of the chemically modified mRNA prepared in this example was evaluated by detecting the content of TGFβ3 protein in cells.
100μg未修饰和100μg修饰的mRNA对293细胞进行转化，24h后收取细胞沉淀，裂解细胞，裂解液进行elisa实验检测TGFβ3蛋白浓度。293 cells were transformed with 100 μg of unmodified and 100 μg of modified mRNA. After 24 hours, the cell pellet was collected, the cells were lysed, and the lysate was subjected to ELISA assay to detect the concentration of TGFβ3 protein.
ELISA检测TGFβ浓度：ELISA detection of TGFβ concentration:
1.标准品稀释：用coating buffer将TGFβ蛋白标准品稀释成1μg/ml，并依次10倍稀释6个梯度，以coating buffer为阴性对照。1. Standard dilution: use coating buffer to dilute the TGFβ protein standard to 1 μg/ml, and dilute 6 gradients 10-fold in sequence. The coating buffer is used as a negative control.
2.包被：取96孔板依次加入7个浓度梯度的标准品溶液、细胞裂解液、阴性对照，100μl/孔，平行2孔。2～8℃封闭过夜。2. Coating: Take a 96-well plate and add 7 concentration gradients of standard solution, cell lysate, and negative control in sequence, 100 μl/well, 2 wells in parallel. Block overnight at 2-8°C.
3.洗涤液(1×)配制：用灭菌注射用水将50×的Washing buffer稀释50倍，混匀，即得。3. Preparation of washing solution (1×): Dilute 50× Washing buffer 50 times with sterile water for injection, and mix well.
4.洗板：从4℃冰箱取出包被好的96孔板，将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。4. Wash the plate: Take out the coated 96-well plate from the refrigerator at 4°C, pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add 300 μl of washing solution (1×) to each well, and let it stand for a while. Set for 30s, pour the liquid in the wells into the waste liquid tank, and repeat the plate washing 4 times to pat the wells dry.
5.封闭：加入Blocking buffer，250μl/孔，封上封板膜，室温封闭2h。5. Blocking: Add Blocking buffer, 250μl/well, seal with sealing film, and block at room temperature for 2h.
6.洗板：将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入约300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。6. Wash the plate: pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 μl of washing solution (1×) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
7.用dilution buffer将兔抗TGFβ3一抗稀释1000倍，加入200μl，封上封板膜，室温孵育1.5h。7. Dilute rabbit anti-TGFβ3 primary antibody by 1000 times with dilution buffer, add 200 μl, seal with sealing film, and incubate at room temperature for 1.5h.
8.洗板：将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入约300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。8. Wash the plate: pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 μl of washing solution (1×) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
9.用dilution buffer将Goat pAb to rabbit IgG(HRP)稀释10000倍，200μl/孔，封上封板膜，室温孵育1h。9. Dilute Goat pAb to rabbit IgG(HRP) 10,000 times with dilution buffer, 200 μl/well, cover with sealing film, and incubate at room temperature for 1 h.
10.洗板：将板孔内液体倾倒于废液缸中，在吸水纸上拍干，每孔加入约300μl的洗涤液(1×)，静置30s，将板孔内液体倾倒于废液缸中，如此重复洗板4次拍干板孔。10. Wash the plate: pour the liquid in the plate well into the waste liquid tank, pat dry on absorbent paper, add about 300 μl of washing solution (1×) to each well, let it stand for 30s, and pour the liquid in the plate well into the waste liquid In the tank, repeat the plate washing 4 times and pat the plate wells dry.
11.加TMB buffer，100μl/孔。混匀，置室温避光孵育20min。11. Add TMB buffer, 100μl/well. Mix well and incubate at room temperature for 20 min in the dark.
12.孵育结束后，每孔加入Stop buffer 100μl。12. After the incubation, add 100 μl of Stop buffer to each well.
13.于450nn波长下测定吸光度。13. Measure the absorbance at a wavelength of 450 nm.
14.数据处理：以标准品的平均OD值为纵坐标，浓度为横坐标，采用四参数逻辑拟合方程。14. Data processing: Take the average OD value of the standard product as the ordinate and the concentration as the abscissa, and use a four-parameter logistic fitting equation.
实验结果如图88所示，本发明所示的化学修饰碱基能够显著增加TGFβ3在细胞内的表达效率。The experimental results are shown in Figure 88. The chemically modified bases shown in the present invention can significantly increase the expression efficiency of TGFβ3 in cells.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (20)

  1. 一种修饰的核酸，包括尿嘧啶核苷、胞嘧啶核苷、腺嘌呤核苷和鸟嘌呤核苷，其特征在于，还包括化学修饰的核苷；所述化学修饰的核苷包括化学修饰的尿嘧啶核苷、化学修饰的胞嘧啶核苷、化学修饰的腺嘌呤核苷和化学修饰的鸟嘌呤核苷中的一种或几种。A modified nucleic acid comprising uridine, cytosine, adenosine and guanosine, and characterized in that it also includes chemically modified nucleosides; the chemically modified nucleosides include chemically modified nucleosides. One or more of uridine, chemically modified cytosine, chemically modified adenosine and chemically modified guanosine.
  2. 一种修饰的核酸，其特征在于，由化学修饰的尿嘧啶核苷、化学修饰的胞嘧啶核苷、化学修饰的腺嘌呤核苷和化学修饰的鸟嘌呤核苷组成。A modified nucleic acid is characterized in that it is composed of chemically modified uridine, chemically modified cytosine, chemically modified adenosine and chemically modified guanosine.
  3. 根据权利要求1或2所述修饰的核酸，其特征在于，修饰的核酸为修饰的核糖核酸。The modified nucleic acid according to claim 1 or 2, wherein the modified nucleic acid is a modified ribonucleic acid.
  4. 根据权利要求3所述修饰的核酸，其特征在于，所述化学修饰包括同分异构和/或基团取代；所述基团取代包括甲基取代、甲氧基取代、卤代和N4-乙酰基取代中的一种或几种。The modified nucleic acid according to claim 3, wherein the chemical modification includes isomerization and/or group substitution; and the group substitution includes methyl substitution, methoxy substitution, halogen substitution and N4- One or more of acetyl substitutions.
  5. 根据权利要求3所述修饰的核酸，其特征在于，所述化学修饰的尿嘧啶核苷选自2-氟-2-脱氧尿苷、假尿苷、N1-甲基-假尿苷、5-甲氧基尿苷、2-氟-2-脱氧-假尿苷、2-氟-2-脱氧-N1-甲基-假尿苷和2-氟-2-脱氧-5-甲氧基尿苷中的一种或几种。The modified nucleic acid according to claim 3, wherein the chemically modified uridine is selected from the group consisting of 2-fluoro-2-deoxyuridine, pseudouridine, N1-methyl-pseudouridine, 5- Methoxyuridine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine and 2-fluoro-2-deoxy-5-methoxyuridine one or more of them.
  6. 根据权利要求3所述修饰的核酸，其特征在于，所述化学修饰的胞嘧啶核苷选自N4-乙酰基胞苷、2-氟-2-脱氧胞苷、5-甲基胞苷、2-氟-2-脱氧-5-甲基胞苷和2-氟-2-脱氧-N4-乙酰基胞苷中的一种或几种。The modified nucleic acid according to claim 3, wherein the chemically modified cytidine is selected from the group consisting of N4-acetylcytidine, 2-fluoro-2-deoxycytidine, 5-methylcytidine, 2 -One or more of fluoro-2-deoxy-5-methylcytidine and 2-fluoro-2-deoxy-N4-acetylcytidine.
  7. 根据权利要求3所述修饰的核酸，其特征在于，所述化学修饰的腺嘌呤核苷选自N6-甲基腺苷、2-氟-2-脱氧腺苷和2-氟-2-脱氧-N6-甲基腺苷中的一种或几种。The modified nucleic acid according to claim 3, wherein the chemically modified adenosine is selected from the group consisting of N6-methyladenosine, 2-fluoro-2-deoxyadenosine and 2-fluoro-2-deoxy- One or more of N6-methyladenosine.
  8. 根据权利要求3所述修饰的核酸，其特征在于，所述化学修饰的鸟嘌呤核苷选自2-氟-2-脱氧鸟苷、N7-甲基-鸟苷和2-氟-2-脱氧-N7-甲基-鸟苷中的一种或几种。The modified nucleic acid according to claim 3, wherein the chemically modified guanosine is selected from the group consisting of 2-fluoro-2-deoxyguanosine, N7-methyl-guanosine and 2-fluoro-2-deoxyguanosine One or more of -N7-methyl-guanosine.
  9. 根据权利要求3所述修饰的核酸，其特征在于，还包括Kozak序列的5'UTR、3'UTR和5'帽子结构。The modified nucleic acid according to claim 3, further comprising the 5'UTR, 3'UTR and 5'cap structure of the Kozak sequence.
  10. 根据权利要求3所述修饰的核酸，其特征在于，还包括聚A尾。The modified nucleic acid according to claim 3, further comprising a poly-A tail.
  11. 根据权利要求9或10所述修饰的核酸，其特征在于，所述修饰的核酸为修饰的mRNA。The modified nucleic acid according to claim 9 or 10, wherein the modified nucleic acid is a modified mRNA.
  12. 根据权利要求11所述修饰的核酸，其特征在于，所述mRNA包括编码病毒刺突蛋白的mRNA。The modified nucleic acid of claim 11, wherein the mRNA comprises mRNA encoding a viral spike protein.
  13. 根据权利要求11所述修饰的核酸，其特征在于，所述mRNA包括编码生物体内蛋白酶和蛋白激素的mRNA。The modified nucleic acid according to claim 11, wherein the mRNA comprises mRNA encoding protease and protein hormone in vivo.
  14. 权利要求1～13任意一项所述修饰的核酸在制备疾病诊断剂和/或治疗剂中的应用。Use of the modified nucleic acid according to any one of claims 1 to 13 in the preparation of a disease diagnostic agent and/or a therapeutic agent.
  15. 权利要求12所述修饰的核酸在制备疫苗中的应用。The application of the modified nucleic acid of claim 12 in the preparation of vaccines.
  16. 一种药物制剂，其特征在于，包括权利要求1～13任意一项所述修饰的核酸和赋形剂。A pharmaceutical preparation, characterized by comprising the modified nucleic acid according to any one of claims 1 to 13 and an excipient.
  17. 根据权利要求16所述的药物制剂，其特征在于，所述赋形剂选自生理盐水、柠檬酸缓冲液和柠檬酸-生理盐水缓冲液中的一种。The pharmaceutical preparation according to claim 16, wherein the excipient is selected from one of physiological saline, citrate buffer and citrate-physiological saline buffer.
  18. 权利要求1～13任意一项所述修饰的核酸在疾病诊断和/或治疗中的应用。Use of the modified nucleic acid according to any one of claims 1 to 13 in disease diagnosis and/or treatment.
  19. 根据权利要求18所述的应用，其特征在于，当所述修饰的核酸应用于疾病的治疗时，将所述修饰的核酸导入体内或将所述修饰的核酸在体内表达。The use according to claim 18, characterized in that, when the modified nucleic acid is used for the treatment of a disease, the modified nucleic acid is introduced into the body or the modified nucleic acid is expressed in the body.
  20. 权利要求12所述修饰的核酸作为疫苗的应用，其特征在于，将所述修饰的核酸导入体内。The use of the modified nucleic acid according to claim 12 as a vaccine, characterized in that the modified nucleic acid is introduced into the body.
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