WO2022036858A1 - 一种新型Cap2结构5'帽子类似物及其制备方法 - Google Patents

一种新型Cap2结构5'帽子类似物及其制备方法 Download PDF

Info

Publication number
WO2022036858A1
WO2022036858A1 PCT/CN2020/124720 CN2020124720W WO2022036858A1 WO 2022036858 A1 WO2022036858 A1 WO 2022036858A1 CN 2020124720 W CN2020124720 W CN 2020124720W WO 2022036858 A1 WO2022036858 A1 WO 2022036858A1
Authority
WO
WIPO (PCT)
Prior art keywords
ppp
methyl
omea
omeg
omeu
Prior art date
Application number
PCT/CN2020/124720
Other languages
English (en)
French (fr)
Inventor
胡勇
张苗苗
洪丹
胡迅
Original Assignee
深圳市瑞吉生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市瑞吉生物科技有限公司 filed Critical 深圳市瑞吉生物科技有限公司
Priority to AU2020464470A priority Critical patent/AU2020464470A1/en
Priority to US18/022,062 priority patent/US20230304058A1/en
Priority to KR1020237009040A priority patent/KR20230050447A/ko
Priority to JP2023512445A priority patent/JP2023538643A/ja
Priority to EP20950062.8A priority patent/EP4202053A1/en
Publication of WO2022036858A1 publication Critical patent/WO2022036858A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/36—Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate

Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to a novel Cap2 structure 5' cap analog and a preparation method thereof.
  • RNA with Cap 0 or Cap 1 there are two methods for synthesizing capped mRNA in vitro.
  • the first is to cap the mRNA with vaccinia virus capping enzyme after transcription to generate RNA with Cap 0 or Cap 1.
  • This capping method is very efficient. But the output is erratic and the price is expensive.
  • the enzymatic capping method cannot generate caps of Cap 2 and m6Am.
  • the second method is to add an excess of cap analog to capping during transcription.
  • the most commonly used cap analog is ARCA, which produces an immunogenic Cap 0, but the capping efficiency is only 70%.
  • the yield of this method is also relatively low, at most 1.5 mg of RNA can be prepared per milliliter of transcription reaction system, and the transcription product has high immunogenicity.
  • Cleancap belongs to Cap 1. Unlike ARCA, which uses a dimer (m7GpppG) to initiate T7 transcription, CleanCap uses a trimer (m7GpppAmG) to initiate T7 transcription.
  • the yield of this method is relatively high, 4 mg of capped RNA is prepared per milliliter of transcription reaction system, the capping efficiency can reach 90%, and the immunogenicity of the transcription product is lower than that of ARCA.
  • the present invention provides a novel Cap2 structure 5' cap analog and a preparation method thereof.
  • the novel Cap2 structure 5' cap analog provided by the present invention has higher synthesis efficiency, higher capping efficiency, lower immunogenicity and higher protein translation efficiency.
  • a novel Cap2 structure 5' cap analog the molecular formula of the novel Cap2 structure 5' cap analog is selected from any of the following;
  • the present invention also provides the preparation method of the novel Cap2 structure 5' cap analog described in the above technical solution, comprising the following steps:
  • step 2) Dissolve 2'-O-Methyl-ADP, 2'-O-Methyl-GDP, 2'-O-Methyl-CDP and 2'-O-Methyl-UDP obtained in step 1) in RNA-free
  • the enzyme water was then mixed with 7-Methylguanosine, 7-Methyl-3'-O-Methylguanosine, guanosyltransferase, and 2 ⁇ Reaction Buffer and incubated to obtain m7G(5')ppp(5')(2'OMeA /G/C/U) and 3'-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U);
  • the moles of 2'-O-Methyl-ATP, 2'-O-Methyl-GTP, 2'-O-Methyl-CTP and 2'-O-Methyl-UTP are the same as those of RNase-free
  • the volume ratio of water is 1-30 mmol: 14 ⁇ L.
  • the volume ratio of the step 1) RNase-free water to phosphohydrolase and 2 ⁇ Reaction Buffer is 14:1:15.
  • the enzymatic activity of the phosphohydrolase is 50000U;
  • the components of the 2 ⁇ Reaction Buffer include: 50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM DTT, pH 8.
  • the incubation temperature in the steps 1) to 3) is 37° C.
  • the incubation time is 1 h, respectively.
  • the volume ratio of the step 2) RNase-free water to 7-Methylguanosine, 7-Methyl-3'-O-Methylguanosine, guanosyltransferase, and 2 ⁇ Reaction Buffer is 11:10:10:1:22 .
  • the volume ratio of the step 3) RNase-free water to T4 RNA Ligase 1 and 2 ⁇ T4 RNA Ligase Reaction Buffer is 11:1:22.
  • the components of the 2 ⁇ T4 RNA Ligase Reaction Buffer include: 60 mM Tris-HCl, 20 mM MgCl 2 , 20 mM DTT and 2 mM ATP.
  • the enzymatic activity of the T4 RNA Ligase 1 is 10000U.
  • the 32 Cap2 structure cap analogs in the present invention have higher synthesis efficiency
  • the 32 Cap2 structure cap analogs in the present invention have higher capping efficiency
  • the 32 Cap2 structural cap analogs in the present invention have lower immunogenicity
  • the 32 Cap2 structural cap analogs in the present invention have higher protein translation efficiency.
  • Figures 1-3 verify the structure and homogeneity of each final product by reversed-phase high performance liquid chromatography (RP-HPLC) at 25°C;
  • Figures 4-7 are the fluorescence titration curves of cap analog binding. As the ligand concentration increased, the change in the curve indicated a weaker binding between eIF4E and the corresponding cap analog. The increase in fluorescence signal with increasing ligand concentration is due to an increase in free cap analog in solution. The fluorescence intensity is expressed as a relative value;
  • Figure 8 shows the inhibitory effect of cap analogs on the translation of globulin mRNA in rabbit reticulocyte lysate; native rabbit globulin mRNA was translated in the rabbit reticulocyte lysate system and detected by incorporating [ 3 H]Leu into the protein globulin synthesis;
  • Fig. 9 is the structural formula of the cap structure analog provided by the present invention.
  • Figure 10 shows that the 32 novel Cap2 cap structure analogs provided by the present invention are applied to mRNA synthesis to effectively increase the mRNA synthesis efficiency.
  • the synthetic product (4-6 mg) using the novel Cap2 cap structure analog is significantly higher In ARCA (1.5mg) and Cleancap (3.8mg);
  • Figure 11 shows that 32 new Cap2 cap structure analogs provided by the present invention are applied to mRNA synthesis to effectively increase the capping efficiency of mRNA. 70% of ARCA and 90% of Cleancap;
  • Figure 12 shows that the 32 novel Cap2 cap structure analogs provided by the present invention are applied to mRNA synthesis to effectively reduce immunogenicity, and the intracellular immunogenicity of the synthetic product using the novel Cap2 cap structure analog is only the same as that of the ACRA structure. 9%-52% of mRNA;
  • Figure 13 shows that the novel Cap2 cap structure analog provided by the present invention is applied to mRNA synthesis to effectively improve the protein translation efficiency, and the mRNA encoding luciferase (Luc) is capped with ARCA, Cleancap and the new Cap2 cap structure analog respectively. According to the expression intensity of the mRNA in mice, it can be found that the Luc mRNA with the novel Cap2 cap analog has the highest expression intensity.
  • the present invention provides a novel Cap2 structure 5' cap analog, and the molecular formula of the novel Cap2 structure 5' cap analog is selected from any one of the following;
  • the commonality of the novel Cap2 structure 5' cap analog is based on methoxy modification of 2'OMe, and methoxy modification of A ⁇ G ⁇ C ⁇ U and m7G nucleosides to 2'OMeA or 2'OMeG or 2'OMeC or 2'OMeU and 3'-O-Me-m7G.
  • Caps of novel Cap2 structures are formed by enzymatic reactions.
  • the present invention also provides the preparation method of the novel Cap2 structure 5' cap analog described in the above technical solution, comprising the following steps:
  • step 2) Dissolve 2'-O-Methyl-ADP, 2'-O-Methyl-GDP, 2'-O-Methyl-CDP and 2'-O-Methyl-UDP obtained in step 1) in RNA-free
  • the enzyme water was then mixed with 7-Methylguanosine, 7-Methyl-3'-O-Methylguanosine, guanosyltransferase, and 2 ⁇ Reaction Buffer and incubated to obtain m7G(5')ppp(5')(2'OMeA /G/C/U) and 3'-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U);
  • 2'-O-Methyl-ATP, 2'-O-Methyl-GTP, 2'-O-Methyl-CTP and 2'-O-Methyl-UTP are respectively dissolved in RNase-free water, and then respectively mixed with Phosphohydrolase and 2 ⁇ Reaction Buffer were mixed and incubated to obtain 2'-O-Methyl-ADP, 2'-O-Methyl-GDP, 2'-O-Methyl-CDP and 2'-O-Methyl-UDP.
  • the 2'-O-Methyl-ATP, 2'-O-Methyl-GTP, 2'-O-Methyl-CTP and 2'-O-Methyl-UTP may use conventional commercial products.
  • the amount of the substances of 2'-O-Methyl-ATP, 2'-O-Methyl-GTP, 2'-O-Methyl-CTP and 2'-O-Methyl-UTP is related to RNase-free
  • the volume ratio of the water is preferably 1-30 mmol: 14 ⁇ L, more preferably 10 mmol: 14 ⁇ L.
  • the present invention does not specifically limit the sources of the 2'-O-Methyl-ATP, 2'-O-Methyl-GTP, 2'-O-Methyl-CTP and 2'-O-Methyl-UTP, and commercially available products are used. Or it can be prepared by conventional methods.
  • the volume ratio of the RNase-free water to phosphohydrolase and 2 ⁇ Reaction Buffer is preferably 14:1:15.
  • the enzymatic activity of the phosphohydrolase is preferably 50,000 U; the components of the 2 ⁇ Reaction Buffer preferably include: 50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM DTT, and the pH is 8.
  • the temperature of the incubation is preferably 37°C, and the incubation time is preferably 1 h.
  • the 2'-O-Methyl-ATP, 2'-O-Methyl-GTP, 2'-O-Methyl-CTP and 2'-O-Methyl-UTP are preferably purified by HPLC and then dissolved in RNase-free water.
  • the obtained 2'-O-Methyl-ADP, 2'-O-Methyl-GDP, 2'-O-Methyl-CDP and 2'-O-Methyl-UDP are respectively dissolved in RNase-free water, and then respectively Mixed with 7-Methylguanosine, 7-Methyl-3'-O-Methylguanosine, guanosyltransferase, and 2 ⁇ Reaction Buffer and incubated to obtain m7G(5')ppp(5')(2'OMeA/G/C/ U) and 3'-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U).
  • the volume ratio of the RNase-free water to 7-Methylguanosine, 7-Methyl-3'-O-Methylguanosine, guanosyltransferase and 2 ⁇ Reaction Buffer is preferably 11:10:10:1:22 .
  • the enzymatic activity of the guanosyltransferase is preferably 50000U.
  • the components of the 2 ⁇ Reaction Buffer preferably include: 50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM DTT, and the pH value is 8.
  • the source of the 7-Methylguanosine and 7-Methyl-3'-O-Methylguanosine is not particularly limited in the present invention, and can be prepared by using conventional commercially available or conventional preparation methods.
  • the temperature of the incubation is preferably 37°C, and the incubation time is preferably 1 h.
  • the m7G(5')ppp(5')(2'OMeA/G/C/U) and 3'-O-Me-m7G(5')ppp(5')(2'OMeA/G) obtained by the present invention /C/U) were dissolved in RNase-free water, mixed with T4 RNA Ligase 1, and then mixed with 2'-O-Methyl-ATP, 2'-O-Methyl-GTP, 2'-O-Methyl-CTP Mixed with 2'-O-Methyl-UTP, then mixed with 2 ⁇ T4 RNA Ligase Reaction Buffer and incubated to obtain a new Cap2 structure 5' cap analog.
  • the m7G(5')ppp(5')(2'OMeA/G/C/U) and 3'-O-Me-m7G(5')ppp(5')(2'OMeA /G/C/U) is preferably purified by HPLC and then dissolved in RNase-free water.
  • the volume ratio of the RNase-free water to T4 RNA Ligase 1 and 2 ⁇ T4 RNA Ligase Reaction Buffer is preferably 11:1:22.
  • the enzymatic activity of the T4 RNA ligase 1 is preferably 10000U.
  • the components of the 2 ⁇ T4 RNA Ligase Reaction Buffer preferably include: 60 mM Tris-HCl, 20 mM MgCl 2 , 20 mM DTT and 2 mM ATP.
  • the temperature of the incubation is preferably 37°C, and the incubation time is preferably 1 h.
  • step 3 The solution obtained in step 3 was purified by HPLC and dissolved in RNase-free water for use, with a total volume of 11 ⁇ l.
  • step 5 The solution obtained in step 5 was purified by HPLC and dissolved in RNase-free water as follows:
  • the molecular formula is as follows:
  • Eukaryotic initiation factor 4E is a cap-binding protein that specifically recognizes the cap structure at the 5' end of mRNA, and plays an important role in the initiation of eukaryotic translation. Because eIF4E itself has tryptophan, it can detect fluorescence at 337nm wavelength when excited at 280nm wavelength. After binding to the cap structure, the fluorescence is weakened. Therefore, this method is used as the standard for detecting the binding ability of the cap structure to eIF4E. The results are shown in Figure 4-7.
  • Micrococcal nuclease treatment of rabbit reticulocyte lysates removes nucleic acids and serves as an in vitro translation system for mRNAs in which mRNAs can be expressed.
  • natural rabbit globulin mRNA was added at a final concentration of 5ug/ml, and 3 H-labeled leucine was added at the same time.
  • Micrococcal nuclease was used to treat rabbit reticulocyte lysate to remove nucleic acid, which was used as a reaction system of mRNA in vitro translation system, in which mRNA could be expressed.
  • natural rabbit globulin mRNA was added at a final concentration of 5 ⁇ g/ml, and 3 H-labeled leucine was added at the same time.
  • 100 ⁇ mol of Cap2 cap analogs (ARCA and cleancap were used as controls) were added to each 1 ml reaction system, and the protein in the reaction system was extracted after 24 h, and the content of globulin 3 H was detected.
  • T7 UTP Solution(75mM) 2 ⁇ l Linearized plasmid template ⁇ 8 ⁇ l
  • T7 Enzyme Mix 2 ⁇ l ARCA/Cleancap/Cap2 2 ⁇ l Nuclease-free water to 20 ⁇ l
  • the mRNA molecules with different cap structures obtained in Example 1 are subjected to enzyme digestion, and the ratio of capped and uncapped mRNA molecules can be determined in liquid chromatography mass spectrometry experiments.
  • Beads were centrifuged at 15000g for 5min, the supernatant was removed, 120 ⁇ l of annealed mRNA and tags were added, and incubated at room temperature for 30min, with gentle shaking during the period to ensure sufficient binding;
  • Human PBMC cells were cultured to 10 6 cells/well, and 5 ⁇ g of ARCA, Cleancap and Cap 2 cap mRNA were transfected using Lipofectamine 2000. After 24 h, cells were harvested by trypsinization and resuspended in PBS, centrifuged, and harvested.
  • the fixative was added to the cells, glycine was added for 15 min to stop the fixation, and the cells were collected by centrifugation. Add lysate to resuspend the cells, incubate on ice for 30min, centrifuge at 2400g for 10min at 4°C, and collect the supernatant.
  • TLR3, TLR7, TLR8 and RIG-1 (2-10 ⁇ g) were added to the supernatant (6-10 mg), and incubated at 4°C with gentle agitation for 2 h.
  • ProteinA/G magnetic beads (40 ⁇ L) were added and incubated at 4°C with gentle agitation for 1 h.
  • the magnetic beads were pelleted by centrifugation at 2500 rpm for 30 s, the supernatant was removed, and the magnetic beads were resuspended in 500 ⁇ L RIP buffer. Washing was repeated three times in RIP buffer followed by one wash in PBS.
  • RNA was reverse transcribed (RT) into cDNA, and primers were designed according to the mRNA sequence for quantitative PCR analysis.
  • the mRNA immunogenicity was proportional to the number of mRNA copies precipitated by magnetic beads.
  • the novel Cap2 cap structure analog prepared in Example 1 was used for efficient protein translation efficiency in mRNA synthesis.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
  • Electrotherapy Devices (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Infusion, Injection, And Reservoir Apparatuses (AREA)

Abstract

一种Cap2结构5'帽子类似物及其制备方法，所述Cap2结构5'帽子类似物的分子式选自m7G（5'）ppp（5'）（2'OMeA）p（2'OMeG）；m7G（5'）ppp（5'）（2'OMeG）p（2'OMeG）等任一种。所述Cap2结构5'帽子类似物具有更高的合成效率、更高的加帽效率、更低的免疫原性和更高的蛋白翻译效率。

Description

一种新型Cap2结构5′帽子类似物及其制备方法
本申请要求于2020年08月20日提交中国专利局、申请号为202010842263.2、发明名称为“一种新型Cap2结构5'帽子类似物及其制备方法”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。
技术领域
本发明属于基因工程技术领域，具体涉及一种新型Cap2结构5'帽子类似物及其制备方法。
背景技术
翻译效率和免疫原性是mRNA药物行使功能的两大关键因素。自体免疫蛋白如RIG-I和IFIT识别异常加帽的mRNA，降低了外源mRNA在细胞内的活性和半衰期，使得mRNA药物在体内难以发挥作用。因此，优化mRNA帽结构对于提高mRNA的生物活性和降低免疫原性是至关重要的。
目前在体外合成带帽的mRNA有两种方法，第一种是mRNA转录后使用牛痘病毒加帽酶对其进行加帽，产生带Cap 0或Cap 1的RNA，这种加帽方法很高效，但是产量不稳定，价格也很昂贵。此外酶促加帽法不能产生Cap 2和m6Am的帽子。第二种方法是转录过程中加入过量的帽结构类似物来进行加帽。截至目前，最常用的帽结构类似物是ARCA，使用ARCA可产生具有免疫原性的Cap 0，但是加帽效率只有70％。该方法的产量也比较低，每毫升转录反应体系最多制备1.5mgRNA，转录产物具有很高的免疫原性。另一种叫做Cleancap的新型共转录加帽的方法也被广泛采用。Cleancap属于Cap 1，与ARCA使用二聚体(m7GpppG)启动T7转录不同，CleanCap使用三聚体(m7GpppAmG)启动T7转录。该方法的产量比较高，每毫升转录反应体系制备4mg加帽的RNA，加帽效率可达90％，其转录产物的免疫原性低于ARCA。
基于现有mRNA合成体系中的帽结构类似物的加帽率低，单位合成体积内mRNA产量低以及免疫原性高等缺点，从而研发了新型Cap2帽结 构类似物，既弥补了以上应用上的缺点，又大大提升了蛋白翻译效率。
发明内容
基于上述原因，本发明提供一种新型Cap2结构5'帽子类似物及其制备方法，本发明提供的新型Cap2结构5'帽子类似物具有更高的合成效率、更高的加帽效率、更低的免疫原性和更高的蛋白翻译效率。
本发明通过下述技术方案实现的：
一种新型Cap2结构5'帽子类似物，所述新型Cap2结构5'帽子类似物的分子式选自以下任一种；
m7G(5')ppp(5')(2'OMeA)p(2'OMeG)；
m7G(5')ppp(5')(2'OMeG)p(2'OMeG)；
m7G(5')ppp(5')(2'OMeC)p(2'OMeG)；
m7G(5')ppp(5')(2'OMeU)p(2'OMeG)；
m7G(5')ppp(5')(2'OMeA)p(2'OMeA)；
m7G(5')ppp(5')(2'OMeG)p(2'OMeA)；
m7G(5')ppp(5')(2'OMeC)p(2'OMeA)；
m7G(5')ppp(5')(2'OMeU)p(2'OMeA)；
m7G(5')ppp(5')(2'OMeA)p(2'OMeC)；
m7G(5')ppp(5')(2'OMeG)p(2'OMeC)；
m7G(5')ppp(5')(2'OMeC)p(2'OMeC)；
m7G(5')ppp(5')(2'OMeU)p(2'OMeC)；
m7G(5')ppp(5')(2'OMeA)p(2'OMeU)；
m7G(5')ppp(5')(2'OMeG)p(2'OMeU)；
m7G(5')ppp(5')(2'OMeC)p(2'OMeU)；
m7G(5')ppp(5')(2'OMeU)p(2'OMeU)；
3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeG)；
3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeG)；
3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeG)；
3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeG)；
3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeA)；
3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeA)；
3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeA)；
3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeA)；
3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeC)；
3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeC)；
3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeC)；
3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeC)；
3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeU)；
3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeU)；
3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeU)；
3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeU)。
本发明还提供了上述技术方案所述的新型Cap2结构5'帽子类似物的制备方法，包括以下步骤：
1)将2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP分别溶解在无RNA酶的水中，再分别与磷酸水解酶、2×Reaction Buffer混合后进行孵育，得到2'-O-Methyl-ADP、2'-O-Methyl-GDP、2'-O-Methyl-CDP和2'-O-Methyl-UDP；
2)将所述步骤1)得到的2'-O-Methyl-ADP、2'-O-Methyl-GDP、2'-O-Methyl-CDP和2'-O-Methyl-UDP分别溶解在无RNA酶水中，再分别与7-Methylguanosine、7-Methyl-3'-O-Methylguanosine、鸟苷转移酶、2×Reaction Buffer混合后进行孵育，得到m7G(5')ppp(5')(2'OMeA/G/C/U)和3′-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U)；
3)将所述步骤2)得到的m7G(5')ppp(5')(2'OMeA/G/C/U)和3′-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U)分别溶解在无RNA酶水中，与T4 RNALigase1混合后，再分别与2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP混合，再与2×T4 RNA Ligase Reaction Buffer混合后进行孵育，得到新型Cap2结构5'帽子类似物。
优选的，所述步骤1)2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP的摩尔与无RNA酶的水的体积比均为1～30mmol:14μL。
优选的，所述步骤1)无RNA酶的水与磷酸水解酶、2×Reaction Buffer 的体积比为14:1:15。
优选的，所述磷酸水解酶的酶活为50000U；
所述2×Reaction Buffer的组分包括：50mM Tris-HCl、5mM KCl、1mM MgCl 2、1mM DTT，pH值为8。
优选的，所述步骤1)～3)孵育的温度分别为37℃，所述孵育的时间分别为1h。
优选的，所述步骤2)无RNA酶水与7-Methylguanosine、7-Methyl-3'-O-Methylguanosine、鸟苷转移酶、2×Reaction Buffer的体积比为11:10:10:1:22。
优选的，所述步骤3)无RNA酶水与T4 RNA Ligase 1、2×T4 RNA Ligase Reaction Buffer的体积比为11:1:22。
优选的，所述2×T4 RNA Ligase Reaction Buffer的组分包括：60mM Tris-HCl、20mM MgCl 2、20mM DTT和2mM ATP。
优选的，所述T4 RNA Ligase 1的酶活为10000U。
本发明的有益效果：
与现有帽结构类似物ARCA和Cleancap相比，本发明中的32种Cap2结构帽类似物具有更高的合成效率；
与现有帽结构类似物ARCA和Cleancap相比，本发明中的32种Cap2结构帽类似物具有更高的加帽效率；
与现有帽结构类似物ARCA和Cleancap相比，本发明中的32种Cap2结构帽类似物具有更低的免疫原性；
与现有帽结构类似物ARCA和Cleancap相比，本发明中的32种Cap2结构帽类似物具有更高的蛋白翻译效率。
附图说明
图1-3用反相高效液相色谱(RP-HPLC)在25℃的条件下对各最终产物的结构和均一性进行验证；
图4-7为cap analog结合的荧光滴定曲线。随着配体浓度的升高，曲线的变化表明eIF4E和相应的cap analog之间的结合较弱。随着配体浓度 的增加，荧光信号的增加是由于溶液中游离cap analog的增加。荧光强度以相对值表示；
图8为帽结构类似物对兔网织红细胞裂解液球蛋白mRNA翻译的抑制作用；在兔网织红细胞裂解系统中翻译天然兔球蛋白mRNA，并通过将[ 3H]Leu掺入蛋白中检测球蛋白合成；
图9为本发明所提供的帽结构类似物的结构通式；
图10为本发明提供的32种新型Cap2帽结构类似物应用于mRNA合成有效增加mRNA的合成效率，在每毫升合成体系中，采用新型Cap2帽结构类似物的合成产物(4～6mg)明显高于ARCA(1.5mg)和Cleancap(3.8mg)；
图11为本发明提供的32种新型Cap2帽结构类似物应用于mRNA合成有效增加mRNA的加帽效率，在同等合成条件下，采用新型Cap2帽结构类似物的合成产物95％被加帽，高于ARCA的70％和Cleancap的90％；
图12为本发明提供的的32种新型Cap2帽结构类似物应用于mRNA合成有效降低免疫源性，采用新型Cap2帽结构类似物的合成产物在细胞内的免疫原性仅为采用ACRA结构的相同mRNA的9％-52％；
图13为本发明提供的新型Cap2帽结构类似物应用于mRNA合成有效提高蛋白翻译效率，编码荧光素酶(Luc)的mRNA分别采用ARCA、Cleancap和新型Cap2帽结构类似物进行加帽，对比三种mRNA在小鼠体内的表达强度，可以发现采用新型Cap2帽结构类似物的Luc mRNA表达强度最高。
具体实施方式
本发明提供了一种新型Cap2结构5'帽子类似物，所述新型Cap2结构5'帽子类似物的分子式选自以下任一种；
m7G(5')ppp(5')(2'OMeA)p(2'OMeG)；
m7G(5')ppp(5')(2'OMeG)p(2'OMeG)；
m7G(5')ppp(5')(2'OMeC)p(2'OMeG)；
m7G(5')ppp(5')(2'OMeU)p(2'OMeG)；
m7G(5')ppp(5')(2'OMeA)p(2'OMeA)；
m7G(5')ppp(5')(2'OMeG)p(2'OMeA)；
m7G(5')ppp(5')(2'OMeC)p(2'OMeA)；
m7G(5')ppp(5')(2'OMeU)p(2'OMeA)；
m7G(5')ppp(5')(2'OMeA)p(2'OMeC)；
m7G(5')ppp(5')(2'OMeG)p(2'OMeC)；
m7G(5')ppp(5')(2'OMeC)p(2'OMeC)；
m7G(5')ppp(5')(2'OMeU)p(2'OMeC)；
m7G(5')ppp(5')(2'OMeA)p(2'OMeU)；
m7G(5')ppp(5')(2'OMeG)p(2'OMeU)；
m7G(5')ppp(5')(2'OMeC)p(2'OMeU)；
m7G(5')ppp(5')(2'OMeU)p(2'OMeU)；
3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeG)；
3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeG)；
3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeG)；
3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeG)；
3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeA)；
3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeA)；
3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeA)；
3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeA)；
3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeC)；
3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeC)；
3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeC)；
3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeC)；
3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeU)；
3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeU)；
3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeU)；
3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeU)。
在本发明中，所述新型Cap2结构5'帽子类似物共性是基于甲氧基修饰2'OMe，对A\G\C\U以及m7G核苷进行甲氧基修饰2'OMeA或2'OMeG或2'OMeC或2'OMeU和3′-O-Me-m7G。通过酶学反应形成新型Cap2结构的帽子。
本发明还提供了上述技术方案所述的新型Cap2结构5'帽子类似物的制备方法，包括以下步骤：
1)将2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP分别溶解在无RNA酶的水中，再分别与磷酸水解酶、2×Reaction Buffer混合后进行孵育，得到2'-O-Methyl-ADP、2'-O-Methyl-GDP、2'-O-Methyl-CDP和2'-O-Methyl-UDP；
2)将所述步骤1)得到的2'-O-Methyl-ADP、2'-O-Methyl-GDP、2'-O-Methyl-CDP和2'-O-Methyl-UDP分别溶解在无RNA酶水中，再分别与7-Methylguanosine、7-Methyl-3'-O-Methylguanosine、鸟苷转移酶、2×Reaction Buffer混合后进行孵育，得到m7G(5')ppp(5')(2'OMeA/G/C/U)和3′-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U)；
3)将所述步骤2)得到的m7G(5')ppp(5')(2'OMeA/G/C/U)和3′-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U)分别溶解在无RNA酶水中，与T4 RNA Ligase1混合后，再分别与2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP混合，再与2×T4 RNA Ligase Reaction Buffer混合后进行孵育，得到新型Cap2结构5'帽子类似物。
本发明将2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP分别溶解在无RNA酶的水中，再分别与磷酸水解酶、2×Reaction Buffer混合后进行孵育，得到2'-O-Methyl-ADP、2'-O-Methyl-GDP、2'-O-Methyl-CDP和2'-O-Methyl-UDP。在本发明中，所述2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP采用常规市售产品即可。
在本发明中，所述2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP的物质的量与无RNA酶的水的体积比优选均为1～30mmol:14μL，,更优选为10mmol:14μL。本发明对所述2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP的来源没有特殊限定，采用市售产品或者采用常规方法制备即可。在本发明中，所述无RNA酶的水与磷酸水解酶、2×Reaction Buffer的体积比优 选为14:1:15。在本发明中，所述磷酸水解酶的酶活优选为50000U；所述2×Reaction Buffer的组分优选包括：50mM Tris-HCl、5mM KCl、1mM MgCl 2、1mM DTT，pH值为8。在本发明中，所述孵育的温度优选为37℃，所述孵育的时间优选为1h。在本发明中，所述2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP优选经过HPLC纯化后再溶解在无RNA酶水中。
本发明将得到的2'-O-Methyl-ADP、2'-O-Methyl-GDP、2'-O-Methyl-CDP和2'-O-Methyl-UDP分别溶解在无RNA酶水中，再分别与7-Methylguanosine、7-Methyl-3'-O-Methylguanosine、鸟苷转移酶、2×Reaction Buffer混合后进行孵育，得到m7G(5')ppp(5')(2'OMeA/G/C/U)和3′-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U)。
在本发明中，所述无RNA酶水与7-Methylguanosine、7-Methyl-3'-O-Methylguanosine、鸟苷转移酶、2×Reaction Buffer的体积比优选为11:10:10:1:22。在本发明中，所述鸟苷转移酶的酶活优选为50000U。在本发明中，所述2×Reaction Buffer的组分优选包括：50mM Tris-HCl、5mM KCl、1mM MgCl 2、1mM DTT，pH值为8。本发明对所述7-Methylguanosine和7-Methyl-3'-O-Methylguanosine的来源没有特殊限定，采用常规市售或者采用常规制备方法制备即可。在本发明中，所述孵育的温度优选为37℃，所述孵育的时间优选为1h。
本发明将得到的m7G(5')ppp(5')(2'OMeA/G/C/U)和3′-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U)分别溶解在无RNA酶水中，与T4 RNA Ligase 1混合后，再分别与2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP混合，再与2×T4 RNA Ligase Reaction Buffer混合后进行孵育，得到新型Cap2结构5'帽子类似物。
在本发明中，所述m7G(5')ppp(5')(2'OMeA/G/C/U)和3′-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U)优选进行HPLC纯化后再溶解在无RNA酶水中。
在本发明中，所述无RNA酶水与T4 RNA Ligase 1、2×T4 RNA Ligase Reaction Buffer的体积比优选为11:1:22。在本发明中，所述T4 RNA连接 酶1的酶活优选为10000U。在本发明中，所述2×T4 RNA Ligase Reaction Buffer的组分优选包括：60mM Tris-HCl、20mM MgCl 2、20mM DTT和2mM ATP。在本发明中，所述孵育的温度优选为37℃，所述孵育的时间优选为1h。
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。
实施例1
1.将10mmol的2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP或者2'-O-Methyl-UTP分别溶解在无RNA酶的水中，总体积为14μl。加入1μl(50000U)磷酸水解酶和15μl 2×Reaction Buffer(50mM Tris-HCl；5mM KCl；1mM MgCl 2；1mM DTT；pH8)，37℃下孵育1h，得到2'-O-Methyl-ADP、2'-O-Methyl-GDP、2'-O-Methyl-CDP和2'-O-Methyl-UDP。
2.对以上所得2'-O-Methyl-ADP、2'-O-Methyl-GDP、2'-O-Methyl-CDP和2'-O-Methyl-UDP分别进行进行HPLC纯化并溶解在无RNA酶水中，总体积为11μl。
3.将10μl(10mmol)7-Methylguanosine、10μl(10mmol)7-Methyl-3'-O-Methylguanosine和1μl(50000U)鸟苷转移酶加入到步骤2得到的溶液中，加入22μl 2×Reaction Buffer(50mM Tris-HCl；5mM KCl；1mM MgCl 2；1mM DTT；pH8)，在37℃下孵育1h，得到m7G(5')ppp(5')(2'OMeA/G/C/U)或3′-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U)。
4.对步骤3所得的溶液进行HPLC纯化并溶解在无RNA酶水中备用，总体积为11μl。
5.将10μl(10mmol)的2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP或者2'-O-Methyl-UTP分别与1μl(10000U)T4 RNA连接酶1(ssRNA ligase NEB M0437M)加入到步骤4所得溶液中，加入22μl 2×T4 RNA Ligase Reaction Buffer(60mM Tris-HCl(pH 7.8 at 25℃),20mM MgCl 2,20mM DTT and 2mM ATP)，在37℃下孵育1h，得到新型Cap2结构5'帽子类似物，
6.对步骤5所得的溶液进行HPLC纯化并溶解在无RNA酶水中，方法如下：
1)RP HPLC实验参数设定为流速：lml/min(4.6mm内径层析柱)；0.lml/min(2.1mm内径层析柱)；
2)注入100μl样品(0.lmg/ml)(即每个待检测帽子结构类似物为10μg，流速为lml/min时1μg＝100mAU)；对于2.1mm层析柱，加入l0μl浓度为0.lmg/ml的标样蛋白，即每个待检测帽子结构类似物1μg(在流速为0.lml/min时，1μg＝100mAU)；
3)样品的收集，在有盖的聚丙烯微量离心管中收集洗脱下来的峰，盖紧盖子，于-20℃保存。
分子式如下：
1、m7G(5')ppp(5')(2'OMeA)p(2'OMeG)；
2、m7G(5')ppp(5')(2'OMeG)p(2'OMeG)；
3、m7G(5')ppp(5')(2'OMeC)p(2'OMeG)；
4、m7G(5')ppp(5')(2'OMeU)p(2'OMeG)；
5、m7G(5')ppp(5')(2'OMeA)p(2'OMeA)；
6、m7G(5')ppp(5')(2'OMeG)p(2'OMeA)；
7、m7G(5')ppp(5')(2'OMeC)p(2'OMeA)；
8、m7G(5')ppp(5')(2'OMeU)p(2'OMeA)；
9、m7G(5')ppp(5')(2'OMeA)p(2'OMeC)；
10、m7G(5')ppp(5')(2'OMeG)p(2'OMeC)；
11、m7G(5')ppp(5')(2'OMeC)p(2'OMeC)；
12、m7G(5')ppp(5')(2'OMeU)p(2'OMeC)；
13、m7G(5')ppp(5')(2'OMeA)p(2'OMeU)；
14、m7G(5')ppp(5')(2'OMeG)p(2'OMeU)；
15、m7G(5')ppp(5')(2'OMeC)p(2'OMeU)；
16、m7G(5')ppp(5')(2'OMeU)p(2'OMeU)；
17、3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeG)；
18、3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeG)；
19、3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeG)；
20、3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeG)；
21、3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeA)；
22、3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeA)；
23、3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeA)；
24、3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeA)；
25、3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeC)；
26、3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeC)；
27、3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeC)；
28、3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeC)；
29、3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeU)；
30、3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeU)；
31、3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeU)；
32、3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeU)。
制备的32种新型Cap2帽结构类似物在25℃下利用RP HPLC进行鉴定。结果如图1-3所示。
实施例2
通过检测实施例1制备的32种新型Cap2帽结构类似物与eIF4E结合能力检测，结合能力越高翻译活性越强。
帽子结构类似物与eIF4E结合能力检测。真核起始因子4E(eIF4E)是一种帽结合蛋白，可以特异性地识别mRNA的5'端的帽子结构，它在真核翻译的起始过程中发挥重要作用。eIF4E自身因带有色氨酸可以在280nm波长下激发在337nm波长处检测到荧光，在与帽结构结合后，荧光减弱，因此本方法作为检测帽结构与eIF4E结合能力的标准。结果见图4-7。
实验方法：1.4mL 0.1μM的eIF4E蛋白(溶剂为50mM HEPES/KOH(pH7.2),100mM KCl,0.5mM EDTA,1mM DTT)中滴加1μl不同浓度的帽结构溶液，在280nm波长下激发在337nm波长处检测荧光变化情况。
实施例3
通过检测实施例1制备的32种新型Cap2帽结构类似物对球蛋白表 达水平的影响，确定Cap2帽子结构类似物的生物活性。
微球菌核酸酶处理兔网织红细胞裂解物除去核酸，作为mRNA体外翻译系统，mRNA可在其中表达。在该体系中加入天然兔球蛋白mRNA,终浓度为5ug/ml，同时加入 3H标记的亮氨酸。在每1ml反应体系中分别加入100umol Cap2帽子结构类似物(ARCA和cleancap作为对照)，24小时后提取反应体系中的蛋白质，检测球蛋白 3H含量。
由于帽子结构类似物竞争结合eIF4E，加入的帽结构类似物与eIF4E结合能力越强，则加入的天然mRNA翻译水平越低，球蛋白 3H含量则越低。结果见图8。
实验方法：微球菌核酸酶处理兔网织红细胞裂解物除去核酸，作为mRNA体外翻译系统反应体系，mRNA可在其中表达。在该体系中加入天然兔球蛋白mRNA,终浓度为5μg/ml，同时加入 3H标记的亮氨酸。在每1ml反应体系中分别加入100μmol Cap2帽子结构类似物(ARCA和cleancap作为对照)，24h后提取反应体系中的蛋白质，检测球蛋白 3H含量。
实施例4
实施例1制备的32种新型Cap2帽结构类似物应用于mRNA合成有效增加mRNA的合成效率(4-6mg/ml)。
实验方法：
1.合成mRNA前，先用NotI线性化质粒，4℃酶切过夜。
2.DNA模板抽提。
3.体外转录合成mRNA,分别使用ARCA、Cleancap和本发明中32种Cap2作为帽结构，反应体系如表1：
表1 反应体系
组分 用量
T7 10X Reaction Buffer 2μl
T7 ATP Solution(75mM) 2μl
T7 CTP Solution(75mM) 2μl
T7 GTP Solution(75mM) 2μl
T7 UTP Solution(75mM) 2μl
线性化质粒模板 <8μl
T7 Enzyme Mix 2μl
ARCA/Cleancap/Cap2 2μl
Nuclease-free水 至20μl
37℃反应6h。TURBO DNase消化15min。
4.使用LiCl溶液对转录产物进行纯化，计算反应得率。
结果如表2、图10，使用ARCA帽结构时，每毫升转录反应体系最高可制备1.5mg mRNA，使用Cleancap时，每毫升转录反应体系最高可制备3.8mg mRNA，使用本发明中的cap2时，每毫升转录反应体系最高可制备至4～6mg mRNA。
表2 1ml mRNA合成反应体系所得的终产物质量(mg)
ARCA Cleancap 1 2 3 4 5 6 7
1.5 3.8 4.5 4.9 5.8 4.7 4.4 5.1 5.3
8 9 10 11 12 13 14 15 16
5.3 4.8 6.0 4.7 4.9 5.1 5.5 4.6 4.2
17 18 19 20 21 22 23 24 25
4.7 4.1 4.4 4.6 4.8 4.6 4.5 4.7 4.1
26 27 28 29 30 31 32 —— ——
4.6 4.7 4.8 5.3 5.3 4.5 4.3 —— ——
实施例5
实施例1制备的32种新型Cap2帽结构类似物应用于mRNA合成有效增加加帽效率(95-98％)。
实验方法：
将实施例1中的得到的不同帽结构的mRNA分子进行酶切，可在液相色谱质谱分析实验中对加帽和未加帽的mRNA分子比例进行测定。
Beads预处理：
1、使用磁铁浓缩beads，吸去上清存储液，加入等体积的0.1M NaOH+0.05M NaCl；
2、吸去上清，用等体积0.1M NaCl重悬beads，保持浓度不变；
3、mRNA与剪切标签退火，配置以下反应体系。
表3 反应体系
10x RNase H reaction buffer 12μl
RNase H probe 500pmol
mRNA 100pmol
total 120μl
梯度退火：95℃ 5min
          65℃ 2min
          55℃ 2min
          22℃ hold
4、剪切标签与beads结合
Beads经过15000g 5min离心，去上清，加入120μl退火后的mRNA和标签，室温孵育30min，期间轻轻震荡，确保充分结合；
5、RNase H剪切
加入10μl RNase H，37℃孵育3h。用磁石沉淀beads，弃上清。
加入100μl washing buffer，充分混匀，磁石沉淀2min，弃上清。
重复3次。
DI water洗3次，
6、RppH消化
10units RppH in nebuffer 2室温消化1h。
7、DI water洗3次。
8、洗脱
吸去DI water，加入100μl预热至80℃的75％乙醇，温育3min，磁石沉淀3min，取上清，蒸发离心45min，至体积为10μl，重悬至50μl 100M EDTA/1％MeOH，待LC-MS分析。
9、LC-MS分析不同帽结构类似物合成的mRNA加帽率。
结果如表4和图11所示，使用ARCA帽结构时，转录产物加帽率最高为70％，使用Cleancap时，转录产物加帽率最高为90％，，使用本发明中的cap2时，转录产物加帽率最高可达95％。
表4 使用不同帽结构时mRNA加帽率
ARCA Cleancap 1 2 3 4 5 6 7
70％ 90％ 94.91％ 95.1％ 94.82％ 94.72％ 94.88％ 94.65％ 94.76％
8 9 10 11 12 13 14 15 16
94.79％ 94.73％ 94.93％ 95.2％ 94.86％ 94.77％ 94.82％ 94.83％ 94.84％
17 18 19 20 21 22 23 24 25
94.91％ 94.98％ 95％ 94.42％ 94.55％ 94.83％ 94.65％ 94.72％ 94.8％
26 27 28 29 30 31 32 —— ——
94.97％ 94.88％ 94.62％ 94.93％ 94.8％ 94.7％ 94.66％ —— ——
实施例6
实施例1制备的32种新型Cap2帽结构类似物应用于mRNA合成有效降低免疫源性。
实验方法：采用RNA免疫共沉淀的方法，将胞内免疫蛋白TLR3、TLR7、TLR8和RIG-1与其结合的RNA一起进行免疫沉淀，最后对这些mRNA进行实时定量PCR鉴定，其相对丰度与其免疫原性呈正比关系。
实验方法：
1.细胞收集
培养人PBMC细胞至10 6个/孔，使用Lipofectamine 2000将5μg ARCA、Cleancap和Cap 2帽结构的mRNA转染细胞。24h后通过胰蛋白酶消化收集细胞并在PBS中重悬细胞，离心，收集细胞。
2.细胞固定和裂解
在细胞中加入固定液，15min加入甘氨酸终止固定，离心收集细胞。加入裂解液重悬细胞，冰孵育30min，4℃，2400g离心10min，收集上清。
3.RNA免疫沉淀
将TLR3、TLR7、TLR8和RIG-1的抗体(2～10μg)加入上清液中(6～10mg)，4℃轻柔搅动孵育2h。加入proteinA/G磁珠(40μL)，4℃轻柔搅动孵育1h。
4.洗去未结合的物质
2500rpm离心30s沉淀磁珠，移去上清液，在500μL RIP缓冲液中重悬磁珠。RIP缓冲液中重复清洗共三次，随后在PBS中清洗一次。
5.对免疫沉淀后RBP上结合的RNA进行纯化
在TRIzol RNA提取试剂(1mL)中重悬磁珠，分离共沉淀的RNA。使用不含核酸酶的水(20μL)洗脱RNA。
6.将RNA逆转录(RT)为cDNA，并根据mRNA序列设计引物进行定量PCR分析，mRNA免疫原性与磁珠沉淀的mRNA拷贝数成正比。
结果如表5和图12，本发明中的Cap2帽结构mRNA免疫原性明显低于ARCA和Cleancap帽结构的mRNA免疫原性。
表5 使用不同帽结构时mRNA的胞内免疫原性，所有结果为相对于ARCA的相对值；
表5 Cap2帽结构mRNA免疫原性实验结果
ARCA Cleancap 1 2 3 4 5 6 7
1 0.76 0.40 0.37 0.28 0.48 0.52 0.38 0.24
8 9 10 11 12 13 14 15 16
0.31 0.4 0.18 0.31 0.19 0.23 0.35 0.34 0.48
17 18 19 20 21 22 23 24 25
0.44 0.31 0.26 0.51 0.15 0.29 0.41 0.48 0.29
26 27 28 29 30 31 32 —— ——
0.49 0.09 0.52 0.27 0.31 0.4 0.3 —— ——
实施例7
实施例1制备的新型Cap2帽结构类似物应用于mRNA合成有效蛋白翻译效率。
实验方法：将等量编码荧光素酶luciferase的mRNA皮内注射于小鼠背部，24h后尾静脉注射荧光素酶底物，发光强度与有效目的蛋白翻译率成比。
实验结果如图13，本法明中的新型Cap2帽结构类似物应用于mRNA合成有效蛋白翻译效率明显高于ARCA和Cleancap帽结构的蛋白翻译效率。
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。

Claims (10)

  1. 一种新型Cap2结构5'帽子类似物，其特征在于，所述新型Cap2结构5'帽子类似物的分子式选自以下任一种；
    m7G(5')ppp(5')(2'OMeA)p(2'OMeG)；
    m7G(5')ppp(5')(2'OMeG)p(2'OMeG)；
    m7G(5')ppp(5')(2'OMeC)p(2'OMeG)；
    m7G(5')ppp(5')(2'OMeU)p(2'OMeG)；
    m7G(5')ppp(5')(2'OMeA)p(2'OMeA)；
    m7G(5')ppp(5')(2'OMeG)p(2'OMeA)；
    m7G(5')ppp(5')(2'OMeC)p(2'OMeA)；
    m7G(5')ppp(5')(2'OMeU)p(2'OMeA)；
    m7G(5')ppp(5')(2'OMeA)p(2'OMeC)；
    m7G(5')ppp(5')(2'OMeG)p(2'OMeC)；
    m7G(5')ppp(5')(2'OMeC)p(2'OMeC)；
    m7G(5')ppp(5')(2'OMeU)p(2'OMeC)；
    m7G(5')ppp(5')(2'OMeA)p(2'OMeU)；
    m7G(5')ppp(5')(2'OMeG)p(2'OMeU)；
    m7G(5')ppp(5')(2'OMeC)p(2'OMeU)；
    m7G(5')ppp(5')(2'OMeU)p(2'OMeU)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeG)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeG)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeG)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeG)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeA)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeA)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeA)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeA)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeC)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeC)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeC)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeC)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeA)p(2'OMeU)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeG)p(2'OMeU)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeC)p(2'OMeU)；
    3′-O-Me-m7G(5')ppp(5')(2'OMeU)p(2'OMeU)。
  2. 权利要求1所述的新型Cap2结构5'帽子类似物的制备方法，其特征在于，包括以下步骤：
    1)将2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP分别溶解在无RNA酶的水中，再分别与磷酸水解酶、2×Reaction Buffer混合后进行孵育，得到2'-O-Methyl-ADP、2'-O-Methyl-GDP、2'-O-Methyl-CDP和2'-O-Methyl-UDP；
    2)将所述步骤1)得到的2'-O-Methyl-ADP、2'-O-Methyl-GDP、2'-O-Methyl-CDP和2'-O-Methyl-UDP分别溶解在无RNA酶水中，再分别与7-Methylguanosine、7-Methyl-3'-O-Methylguanosine、鸟苷转移酶、2×Reaction Buffer混合后进行孵育，得到m7G(5')ppp(5')(2'OMeA/G/C/U)和3′-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U)；
    3)将所述步骤2)得到的m7G(5')ppp(5')(2'OMeA/G/C/U)和3′-O-Me-m7G(5')ppp(5')(2'OMeA/G/C/U)分别溶解在无RNA酶水中，与T4 RNA Ligase1混合后，再分别与2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP混合，再与2×T4 RNA Ligase Reaction Buffer混合后进行孵育，得到新型Cap2结构5'帽子类似物。
  3. 根据权利要求2所述的制备方法，其特征在于，所述步骤1)2'-O-Methyl-ATP、2'-O-Methyl-GTP、2'-O-Methyl-CTP和2'-O-Methyl-UTP的物质的量与无RNA酶的水的体积比均为1～30mmol:14μL。
  4. 根据权利要求2所述的制备方法，其特征在于，所述步骤1)无RNA酶的水与磷酸水解酶、2×Reaction Buffer的体积比为14:1:15。
  5. 根据权利要求4所述的制备方法，其特征在于，所述磷酸水解酶的酶活为50000U；
    所述2×Reaction Buffer的组分包括：50mM Tris-HCl、5mM KCl、1mM MgCl 2、1mM DTT，pH值为8。
  6. 根据权利要求2所述的制备方法，其特征在于，所述步骤1)～3)孵育的温度分别为37℃，所述孵育的时间分别为1h。
  7. 根据权利要求2所述的制备方法，其特征在于，所述步骤2)无RNA酶水与7-Methylguanosine、7-Methyl-3'-O-Methylguanosine、鸟苷转移酶、2×Reaction Buffer的体积比为11:10:10:1:22。
  8. 根据权利要求3所述的制备方法，其特征在于，所述步骤3)无RNA酶水与T4 RNA Ligase 1、2×T4 RNA Ligase Reaction Buffer的体积比为11:1:22。
  9. 根据权利要求2或8所述的制备方法，其特征在于，所述2×T4 RNA Ligase Reaction Buffer的组分包括：60mM Tris-HCl、20mM MgCl 2、20mM DTT和2mM ATP。
  10. 根据权利要求2或8所述的制备方法，其特征在于，所述T4 RNA Ligase 1的酶活为10000U。
PCT/CN2020/124720 2020-08-20 2020-10-29 一种新型Cap2结构5'帽子类似物及其制备方法 WO2022036858A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU2020464470A AU2020464470A1 (en) 2020-08-20 2020-10-29 Novel 5'cap analog having cap2 structure and preparation method therefor
US18/022,062 US20230304058A1 (en) 2020-08-20 2020-10-29 Novel 5'cap analog having cap2 structure and preparation method therefor
KR1020237009040A KR20230050447A (ko) 2020-08-20 2020-10-29 Cap2 구조를 갖는 신규한 5'Cap 유사체 및 이를 위한 제조방법
JP2023512445A JP2023538643A (ja) 2020-08-20 2020-10-29 Ｃａｐ２構造を有する新規な５’キャップアナログおよびその調製方法
EP20950062.8A EP4202053A1 (en) 2020-08-20 2020-10-29 Novel 5'cap analog having cap2 structure and preparation method therefor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010842263.2A CN111944864B (zh) 2020-08-20 2020-08-20 一种Cap2结构5＇帽子类似物及其制备方法
CN202010842263.2 2020-08-20

Publications (1)

Publication Number Publication Date
WO2022036858A1 true WO2022036858A1 (zh) 2022-02-24

Family

ID=73358694

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/124720 WO2022036858A1 (zh) 2020-08-20 2020-10-29 一种新型Cap2结构5'帽子类似物及其制备方法

Country Status (7)

Country Link
US (1) US20230304058A1 (zh)
EP (1) EP4202053A1 (zh)
JP (1) JP2023538643A (zh)
KR (1) KR20230050447A (zh)
CN (31) CN113337558A (zh)
AU (1) AU2020464470A1 (zh)
WO (1) WO2022036858A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024077351A1 (en) * 2022-10-12 2024-04-18 Pharmorage Pty Limited Modified oligonucleotides
WO2024075022A3 (en) * 2022-10-04 2024-07-25 BioNTech SE Rna constructs and uses thereof
WO2024153222A1 (zh) * 2023-01-20 2024-07-25 深圳瑞吉生物科技有限公司 一种用于核酸的5'端加帽的化合物及其应用
WO2024153245A1 (zh) * 2023-01-19 2024-07-25 广州市恒诺康医药科技有限公司 用于rna加帽的化合物及其应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113603739B (zh) * 2021-08-27 2024-08-23 上海兆维科技发展有限公司 一种加帽类似物及其应用
CN115260264B (zh) * 2022-02-28 2023-06-09 广州市恒诺康医药科技有限公司 用于rna加帽的化合物及其应用
CA3206012A1 (en) 2022-02-28 2023-08-28 Guangzhou Henovcom Bioscience Co., Ltd. Compounds for rna capping and uses thereof
CN114540444B (zh) * 2022-04-23 2022-08-09 江苏申基生物科技有限公司 一种加帽组合物及其制备方法和体外转录反应体系
WO2024051696A1 (zh) * 2022-09-05 2024-03-14 广州市恒诺康医药科技有限公司 用于rna加帽的化合物及其应用
CN118048420B (zh) * 2024-04-16 2024-08-09 楷拓生物科技(苏州)有限公司 一种加帽mRNA的制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102076851A (zh) * 2008-05-02 2011-05-25 Epi中心科技公司 Rna的选择性的5′连接标记
CN108366604A (zh) * 2015-09-21 2018-08-03 垂林克生物技术公司 用于合成5’-加帽rna的组合物和方法

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6312926B1 (en) * 1998-08-14 2001-11-06 University Of Medicine & Dentistry Of New Jersey mRNA capping enzymes and uses thereof
US7074596B2 (en) * 2002-03-25 2006-07-11 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Synthesis and use of anti-reverse mRNA cap analogues
CA2692906C (en) * 2007-06-19 2016-01-19 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Synthesis and use of anti-reverse phosphorothioate analogs of the messenger rna cap
CA2701362A1 (en) * 2007-10-09 2009-04-16 European Molecular Biology Laboratory (Embl) Soluble fragments of influenza virus pb2 protein capable of binding rna-cap
EP3524275A1 (en) * 2009-04-22 2019-08-14 Massachusetts Institute Of Technology Innate immune supression enables repeated delivery of long rna molecules
EP3434774A1 (en) * 2013-01-17 2019-01-30 ModernaTX, Inc. Signal-sensor polynucleotides for the alteration of cellular phenotypes
JP2016515216A (ja) * 2013-03-14 2016-05-26 シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド メッセンジャーｒｎａのキャップ効率の定量的評価
PL415967A1 (pl) * 2016-01-29 2017-07-31 Univ Warszawski 5'-tiofosforanowe analogi końca 5' mRNA (kapu), sposób ich otrzymywania i zastosowanie
WO2018075827A1 (en) * 2016-10-19 2018-04-26 Arcturus Therapeutics, Inc. Trinucleotide mrna cap analogs
US11072808B2 (en) * 2018-10-04 2021-07-27 New England Biolabs, Inc. Methods and compositions for increasing capping efficiency of transcribed RNA
CN113166737A (zh) * 2018-10-04 2021-07-23 新英格兰生物实验室公司 提高转录的rna的加帽效率的方法和组合物
CN110218753A (zh) * 2019-05-27 2019-09-10 湖北爱济莱斯生物科技有限公司 一种提高体外合成mRNA稳定性的方法
CN110331147A (zh) * 2019-06-21 2019-10-15 苏州吉玛基因股份有限公司 一种mRNA的制备方法及其在肿瘤治疗中的应用
CN110760535A (zh) * 2019-10-23 2020-02-07 青岛宁逸生物科技有限公司 一种用于体外转录mRNA的载体及其构建方法、利用载体转录得到mRNA的方法和应用
CN111041025B (zh) * 2019-12-17 2021-06-18 深圳市瑞吉生物科技有限公司 基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子及其制备方法
CN111218458B (zh) * 2020-02-27 2020-11-20 珠海丽凡达生物技术有限公司 编码SARS-CoV-2病毒抗原的mRNA和疫苗及疫苗的制备方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102076851A (zh) * 2008-05-02 2011-05-25 Epi中心科技公司 Rna的选择性的5′连接标记
CN108366604A (zh) * 2015-09-21 2018-08-03 垂林克生物技术公司 用于合成5’-加帽rna的组合物和方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANAND RAMANATHAN, G. BRETT ROBB, SIU-HONG CHAN: "mRNA capping: biological functions and applications", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 44, no. 16, 19 September 2016 (2016-09-19), GB , pages 7511 - 7526, XP055388045, ISSN: 0305-1048, DOI: 10.1093/nar/gkw551 *
FURUICHIP Y, MORGAN M, SHATKIN A J, JELINEKT W, SALDITT-GEORGIEFFT M, DARNELLT J E, DARNELL JAMES E: "Methylated, Blocked 5' Termini in HeLa Cell mRNA (7-methylguanosine/N6-methyladenosine)", PROC. NAT. ACAD. SCI., vol. 72, no. 5, 31 May 1975 (1975-05-31), pages 1904 - 1908, XP055902399 *
KUMAR PARIMAL, SWEENEY TREVOR R., SKABKIN MAXIM A., SKABKINA OLGA V., HELLEN CHRISTOPHER U. T., PESTOVA TATYANA V.: "Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5′-terminal regions of cap0-, cap1- and 5′ppp- mRNAs", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 42, no. 5, 1 March 2014 (2014-03-01), GB , pages 3228 - 3245, XP055902396, ISSN: 0305-1048, DOI: 10.1093/nar/gkt1321 *
WERNER MARIA, PURTA ELŻBIETA, KAMINSKA KATARZYNA H., CYMERMAN IWONA A., CAMPBELL DAVID A., MITTRA BIDYOTTAM, ZAMUDIO JESSE R., STU: "2′-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 39, no. 11, 1 June 2011 (2011-06-01), GB , pages 4756 - 4768, XP055902403, ISSN: 0305-1048, DOI: 10.1093/nar/gkr038 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024075022A3 (en) * 2022-10-04 2024-07-25 BioNTech SE Rna constructs and uses thereof
WO2024077351A1 (en) * 2022-10-12 2024-04-18 Pharmorage Pty Limited Modified oligonucleotides
WO2024153245A1 (zh) * 2023-01-19 2024-07-25 广州市恒诺康医药科技有限公司 用于rna加帽的化合物及其应用
WO2024153222A1 (zh) * 2023-01-20 2024-07-25 深圳瑞吉生物科技有限公司 一种用于核酸的5'端加帽的化合物及其应用

Also Published As

Publication number Publication date
CN113416762A (zh) 2021-09-21
CN113122596A (zh) 2021-07-16
CN113234781A (zh) 2021-08-10
CN113337559A (zh) 2021-09-03
CN113278667A (zh) 2021-08-20
US20230304058A1 (en) 2023-09-28
CN113337560A (zh) 2021-09-03
CN113234780A (zh) 2021-08-10
CN113308503A (zh) 2021-08-27
CN113234777A (zh) 2021-08-10
CN113355381A (zh) 2021-09-07
CN113106135A (zh) 2021-07-13
AU2020464470A9 (en) 2024-06-13
CN113106134A (zh) 2021-07-13
CN113122596B (zh) 2023-01-24
CN113122597A (zh) 2021-07-16
CN113136405A (zh) 2021-07-20
CN113106133A (zh) 2021-07-13
CN113308504A (zh) 2021-08-27
CN111944864B (zh) 2021-06-22
CN113355380A (zh) 2021-09-07
CN113234778A (zh) 2021-08-10
CN113278665A (zh) 2021-08-20
EP4202053A1 (en) 2023-06-28
CN113278666A (zh) 2021-08-20
AU2020464470A1 (en) 2023-05-04
CN111944864A (zh) 2020-11-17
CN113416763A (zh) 2021-09-21
CN113201566A (zh) 2021-08-03
CN113234775A (zh) 2021-08-10
KR20230050447A (ko) 2023-04-14
CN113278668A (zh) 2021-08-20
CN113122595A (zh) 2021-07-16
CN113234779A (zh) 2021-08-10
CN113234782A (zh) 2021-08-10
CN113337561A (zh) 2021-09-03
CN113337558A (zh) 2021-09-03
CN113234776A (zh) 2021-08-10
JP2023538643A (ja) 2023-09-08

Similar Documents

Publication Publication Date Title
WO2022036858A1 (zh) 一种新型Cap2结构5&#39;帽子类似物及其制备方法
JPWO2013118878A1 (ja) 環状ｒｎａ及びタンパク質の製造方法
US20230061936A1 (en) Methods of dosing circular polyribonucleotides
WO2021169980A1 (en) Compositions and methods for detecting nucleic acid-protein interactions
WO2024002269A1 (zh) 蛋白质介导的mRNA靶向分子及其制备方法和应用
Kafasla et al. Association of the 72/74-kDa proteins, members of the heterogeneous nuclear ribonucleoprotein M group, with the pre-mRNA at early stages of spliceosome assembly
WO2022120936A1 (zh) 一种修饰的核酸及其应用
Jensen et al. The FlpTRAP system for purification of specific, endogenous chromatin regions

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20950062

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2023512445

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20237009040

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: AU2020464470

Country of ref document: AU

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020950062

Country of ref document: EP

Effective date: 20230320

ENP Entry into the national phase

Ref document number: 2020464470

Country of ref document: AU

Date of ref document: 20201029

Kind code of ref document: A