WO2023213237A1 - 一种含开环核苷结构的起始加帽寡核苷酸引物 - Google Patents

一种含开环核苷结构的起始加帽寡核苷酸引物 Download PDF

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WO2023213237A1
WO2023213237A1 PCT/CN2023/091598 CN2023091598W WO2023213237A1 WO 2023213237 A1 WO2023213237 A1 WO 2023213237A1 CN 2023091598 W CN2023091598 W CN 2023091598W WO 2023213237 A1 WO2023213237 A1 WO 2023213237A1
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unsubstituted
substituted
nucleoside
open
ring
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缪佳颖
黄磊
沈奇
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江苏申基生物科技有限公司
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Priority to US18/520,609 priority Critical patent/US20240132534A1/en

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    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the technical fields of chemistry and bioengineering, and in particular to an initial capped oligonucleotide primer containing a ring-opening nucleoside structure.
  • mRNAs messenger RNAs
  • capped consisting of a 5'-5' triplet between two nucleoside moieties. Phosphate bonding and the 7-methyl group on the distal guanine ring, capping of the mRNA promotes its normal function in the cell. Synthesis of mRNA through in vitro transcription has become an important tool for introducing foreign genes and expressing proteins, and is widely used in the treatment and prevention of diseases. Synthesis of mRNA through in vitro transcription allows workers to prepare RNA that performs appropriately in various biological applications. molecular.
  • Such applications include research applications and commercial production of polypeptides, e.g., production in cell-free translation systems of polypeptides containing "unnatural" amino acids at specific sites, or production in cultured cells of polypeptides required for their activity or stability. Post-translationally modified peptides. In the latter system, synthesis takes significantly longer and therefore more protein is produced.
  • the in vitro transcription yield of mRNA and the 5' capped analog are key processes in the preparation of mRNA.
  • the system currently used for chemical capping of mRNA cannot achieve high efficiency.
  • Patent CN201680067458.6 reports compositions and methods for synthesizing 5'-capped RNA.
  • the starting capped oligonucleotide primer has the general form m 7 Gppp[N 2,Ome ] n [N] m , where m 7 G is N 7 -methylated guanosine or any guanosine analogue, N is any natural, modified or non-natural nucleoside, "n” can be any integer from 0 to 4 and "m” can be an integer from 1 to 9.
  • Cleancap belongs to Cap1. Unlike ARCA, which uses a dimer (m 7 GpppG) to initiate T7 transcription, CleanCap uses a trimer (m 7 GpppAmG) 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%. The immunogenicity of its transcription products is lower than ARCA.
  • Patent US10968248B2 discloses Trinucleotide mRNA cap analogs, involving trinucleotide cap analogs for improving in vitro mRNA synthesis and m 7 G(5')p 3 -RNA transcription. in this structure The third nucleotide in the cap structure is replaced by open-circuit UNA. Since it is the starting nucleotide of transcription, it is not conducive to the recognition of T7 RNA polymerase, reducing the capping efficiency and also reducing the efficiency of in vitro transcription.
  • the present application provides an initial capped oligonucleotide primer containing an open-loop nucleoside structure.
  • the initial capped oligonucleotide primer containing an open-loop nucleoside structure contains a UNA structure to replace the original five
  • the sugar ring structure after replacement, has good anti-reverse transcription effect during in vitro transcription of mRNA because UNA cannot be used as the starting site of transcription, ultimately allowing the mRNA to achieve higher capping efficiency; the open ring structure of UNA helps The mRNA escapes recognition by the immune system in the body, thus better reducing immunogenicity; at the same time, due to the introduction of unnatural nucleotide UNA, the mRNA is not easily hydrolyzed by ribozymes, increasing the stability of the mRNA in the body after capping.
  • An initial capped oligonucleotide primer containing an open-loop nucleoside structure which includes the following structure:
  • R 1 and R 2 are independently H, OH, alkyl, O-alkyl, and halogen;
  • X 1 , X 2 and X 3 are independently O, CH 2 or NH;
  • Y 1 , Y 2 and Y 3 are independently O, S, Se or BH 3 ;
  • R a and R b are independently
  • R 3 and R 4 are independently hydrogen, hydroxyl, substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted N-di Hydrocarbyl, substituted or unsubstituted O-aryl, substituted or unsubstituted S-aryl, substituted or unsubstituted NH-aryl, substituted or unsubstituted O-aralkyl, substituted or unsubstituted S- Aralkyl, substituted or unsubstituted NH-aralkyl;
  • B 1 and B 2 are independently natural, modified, or non-natural nucleobases.
  • the preparation method of the initial capped oligonucleotide primer containing a ring-opening nucleoside structure includes the following steps: (1) Synthesis of m7UrGDP-Im: starting from guanosine, the sugar ring ring-opening nucleoside is synthesized, and in the ring-opening core On the basis of glycosides, diphosphorylation, N7 methylation, and polyphosphoric acid imidazolization reactions are performed sequentially to synthesize m7UrGDP-Im; (2) Preparation of dinucleotides linked by phosphate bonds: through ring-opening or non-ring-opening The phosphoramidite monomer and the ring-opening or non-ring-opening disubstituted nucleoside monomer are coupled under the action of tetrazole to form the first phosphate ester bond.
  • R 5 and R 6 are independently H, OH, alkyl, O-alkyl, and halogen; B 3 and B 4 are independently natural, modified, or non-natural nucleoside bases.
  • the above-mentioned disubstituted nucleoside monomer is selected from any of them.
  • the preparation method of the initial capped oligonucleotide primer containing the open-circuit nucleoside structure specifically includes the following steps:
  • step (1) Dissolve the m7UrGDP-Im obtained in step (1) in the DMF solution containing MnCl2 , and add it to the DMF solution of the phosphate-linked dinucleotide obtained in step (2), stir the reaction at room temperature, 24 After 1 hour, the reaction was stopped with 0.25M EDTA solution; the mixture was loaded onto a DEAE Sephadex column (30 ⁇ 500 cm). The product was eluted using a linear gradient of TEAB eluent from 0 to 1.0 M. Collect the eluted products with HPLC purity >97%, concentrate the above separation liquid, and then load it into a strong anionic resin.
  • the invention provides an initial capped oligonucleotide primer containing an open-circuit nucleoside structure.
  • the molecular structural formula of the initial capped oligonucleotide primer containing an open-circuit nucleoside structure is m7UNGpppA2'OmepG.
  • the provided initial capped oligonucleotide primers containing open-loop nucleoside structures are suitable for mRNA produced using in vitro co-transcription methods using DNA sequences as templates.
  • the DNA sequences can be derived from or modified from viruses, animals, plants and other species.
  • the mRNA produced by it has higher in vitro transcription efficiency, higher capping efficiency, lower immunogenicity and higher protein translation efficiency.
  • the present invention has the following advantages:
  • the initial capped oligonucleotide primer containing an open-loop nucleoside structure of the present invention has higher synthesis efficiency, higher capping efficiency, and lower immunogenicity. , higher protein translation efficiency.
  • Figure 1 is a diagram showing the detection results of the capping rate of mRNA transcription initiated by the initial capping oligonucleotide primer containing a open-circuit nucleoside structure in Example 1;
  • Figure 2 is a graph showing the detection results of the capping rate of mRNA transcription initiated by the initial capping oligonucleotide primer containing the open-circuit nucleoside structure of Example 2;
  • Figure 3 is a graph showing the detection results of the capping rate of mRNA transcription initiated by the initial capping oligonucleotide primer containing the open-circuit nucleoside structure of Example 3;
  • Figure 4 is a graph showing the detection results of the capping rate of mRNA transcription initiated by the initial capping oligonucleotide primer containing the open-circuit nucleoside structure of Example 4;
  • Figure 5 is a graph showing the detection results of the capping rate of mRNA transcription initiated by the cap analogue of Comparative Example 1;
  • Figure 6 is a graph showing the detection results of the capping rate of mRNA transcription initiated by the cap analogue of Comparative Example 2;
  • Figure 7 is a cell phenotype diagram of Examples 1-4 and Comparative Examples 1-2;
  • Figure 8 is a graph showing fluorescence statistical results of Examples 1-4 and Comparative Examples 1-2.
  • the m7UrGDP-Im(J) used in the following examples was prepared through the following steps:
  • the synthesis route of AGP used in Synthesis Example 1 is as follows: weigh 5kg of 2'OMe-rA phosphoramidite monomer in a single-neck bottle, dissolve it in 50L of methylene chloride, and then add 2.73kg of 2',3'acetylguanosine. Lower the temperature to 25 ⁇ 2°C, add 880g of tetrazole under nitrogen blowing, and raise the temperature to 25 ⁇ 2°C for reaction. After the monitoring reaction is completed, add 1.2eq of iodopyridine solution to the reaction solution. After the monitoring reaction is completed, spin it dry. Dissolve the concentrated ointment in 4L dichloromethane. Add 1.1eq of trifluoroacetic acid.
  • the synthesis method of A-UrG-P used in Synthesis Example 2 refers to the AGP synthesis method and the reaction route of A-UrG-P in Example 1, as shown in the following equation (3):
  • the synthesis of D refers to the synthesis steps of intermediate J; the preparation of E2 includes the following steps: weigh 20g of compound D and dissolve it in acetonitrile, add 3eq of triethylamine, cool the reaction solution to 4°C, slowly add acetic anhydride dropwise, and react After completion, add 2eq of TBAF, remove the TBS protecting group and spin to dry column chromatography to obtain compound E2; compound E2 substitutes disubstituted guanosine to obtain A-UrG-P.
  • the synthesis method of UrA-GP used in Synthesis Example 3 refers to the AGP synthesis method in Example 1, and the reaction route flow of UrA-GP is as shown in the following equation (4),
  • the synthesis of D refers to the synthesis steps of intermediate J; the preparation of F4 includes the following steps: (1) Weigh 10g of compound D and dissolve it in DMF, ice bath, slowly add 1.2eq of NaH, and stir at low temperature After 2h, slowly add 2eq of methyl iodide dropwise, react at room temperature for three hours, add water to quench the reaction, filter to obtain the crude product of compound F1, and purify by reverse chromatography; (2) Weigh 2g of compound F and disperse it in 30mL of methanol, add 2eq TBAF, the reaction ends after 2 hours, spin it dry and directly react in the next step; dissolve the spin-dry solid in 30 ml of DCM, add 1.2eq of triethylamine, stir in an ice bath for 20 min, slowly add DMTr-Cl DCM solution, After the dropwise addition, the reaction was completed for half an hour, and the target compound F3 was obtained by column chromatography; (3) Weigh 3g of compound F3, transfer protection by
  • UrA-UrG-P used in Synthesis Example 4 refers to the AGP synthesis method in Example 1.
  • UrA-UrG-P is obtained by the reaction of E2 and F4.
  • the reaction route is as follows: Equation (5):
  • Example 1 Synthesis method of initial capped oligonucleotide primer containing a ring-open nucleoside structure in which both Ra and Rb are five-membered sugar rings
  • m7UrGDP-Im(J) and AGP were synthesized through the following steps: dissolve m7UrGDP-Im(J) (2mol) in a DMF solution containing MnCl 2 (0.2mol), and add to the DMF of AGP (1.8mol) in solution. The reaction was stirred at room temperature. After 24 hours, the reaction was stopped with 10 L of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column (30 x 500 cm). The product was eluted using a linear gradient of TEAB eluent from 0 to 1.0 M. Collect the eluted products with HPLC purity >97%, concentrate the above separation liquid, and then load it into a strong anionic resin.
  • Example 2 Initial capped oligonucleotide primer containing an open-loop nucleoside structure in which Ra is a five-membered sugar ring and Rb is an open-ring structure.
  • the initial capped oligonucleotide primer containing the open-loop nucleoside structure in this example uses m7UrGDP-Im(J) and A-UrG-P as raw materials and is obtained by referring to the synthesis method of the target product in Example 1.
  • Example 3 Initial capped oligonucleotide primer containing an open-ring nucleoside structure in which Ra is an open-ring structure and Rb is a five-membered sugar ring.
  • the initial capped oligonucleotide primer containing the open-loop nucleoside structure in this example uses m7UrGDP-Im(J) and UrA-G-P as raw materials and is obtained by referring to the synthesis method of the target product in Example 1.
  • Example 4 Initial capped oligonucleotide primer containing an open-circle nucleoside structure in which both Ra and Rb are open-circle structures
  • the initial capped oligonucleotide primer containing the open-loop nucleoside structure in this example uses m7UrGDP-Im(J) and UrA-UrG-P as raw materials and is obtained by referring to the synthesis method of the target product in Example 1.
  • the synthesis method of m7 GpppA 2'Ome pG refers to the synthesis method and reaction route of the above examples, as shown in the following equation (7):
  • Test Example 1 Determination of mRNA in vitro transcription yield and capping efficiency
  • the initial capped oligonucleotide primer containing the open-loop nucleoside structure for in vitro synthesis of mRNA first use NotI to linearize the plasmid and digest it overnight at 4°C; DNA template extraction; in vitro transcription and synthesis of mRNA, respectively using the examples 1-4 contains the initial capped oligonucleotide primer of the open-loop nucleoside structure and the capped analog of Comparative Example 1-2 as the cap structure.
  • Liquid chromatography mass spectrometry is used to detect the IVT capping rate of mRNA with different starting cap analogs; first, it is necessary to design a labeled DNA probe that matches the starting base of the transcript product mRNA, usually The label is biotin. Wash the streptavidin-labeled magnetic beads and incubate them with the synthesized DNA probe, mRNA and 10 ⁇ RNase H reaction buffer at room temperature for 30 minutes. Mix slowly while incubating, and then add 20ul RNase. H (5U/ul) was incubated at 37°C for 3h, and mixed every half hour. After the incubation, the magnetic beads are washed.
  • LC-MS Liquid chromatography mass spectrometry
  • RNA capping After the cleaning, 100 ⁇ L of 75% methanol heated to 80°C is added to the magnetic beads. The mixture is heated to 80°C on the hot plate and kept for 3 minutes. Then it is placed on a magnetic stand to absorb the supernatant. Evaporate in a centrifuge and dry at room temperature for 45 min to 10 ⁇ l. The sample was then resuspended in 50 ⁇ l of 100 ⁇ M EDTA/1% MeOH and ready for LC-MS analysis to determine RNA capping during the transcription reaction. Since there is a significant difference in molecular weight between capped and uncapped bases, the difference in molecular mass can be used to determine the capping rate of mRNA transcription initiated by different cap analogues.
  • the sequence of the enzyme digested template used in the test sample template in Figure 1-3 is m7G-pppAGGCGCCACCAUGGUGAGCA (the obtained molecular weight is around 6977), and the test in Figure 4-6
  • the sequence after digestion of the template used in the sample is m7G-pppGGGCGCCACCAUGGUGAGCAA (the obtained molecular weight is around 7322).
  • Test Example 2 Determination of the binding ability of mRNA to RIG I
  • RIG-I mainly includes two repeated caspase activation and recruitment domains (CARD) at the N-terminus, a helicase structure and a C-terminal RNA domain in the middle.
  • CARD caspase activation and recruitment domains
  • the overexpression domain of the N-terminal CARD domain of RIG-I can promote cells to secrete type I interferon (IFN) even in the absence of viral infection. Therefore, this domain is mainly responsible for transmitting signals downstream.
  • IFN interferon
  • 293T cells will be transfected into 293T cells using the initial capped oligonucleotide primers containing open-loop nucleoside structures in Examples 1-4 and the cap analogues in Comparative Examples 1-2 to initiate in vitro transcription of eGFP.
  • the cells were collected after 24 hours, and the intracellular protein RIG-I and its bound RNA were co-immunoprecipitated using the RNA co-immunoprecipitation method. Finally, reverse transcription and real-time quantitative PCR were performed on these mRNAs.
  • the specific cell culture conditions are the same as above. Collect cells 24 hours after transfection. First add fixative and incubate. After 10 minutes, add glycine solution of appropriate concentration to terminate the reaction and collect cells. Use lysis solution to lyse the collected cells, centrifuge at 12,000 rpm and 4°C for 10 min, take the supernatant and incubate with RIG-I or IgG antibodies on a shaker at 4 degrees overnight. Then add 20 ⁇ l Protein A/G magnetic beads, incubate at 4°C for 4 hours, and wash on a magnetic stand. After washing, RNA is extracted and used for subsequent RT-qPCR to verify the expression results.
  • the results of the binding ability of different cap analog nucleotide mRNAs to RIG-1 are as follows in Table 5:
  • Test example 3 Cell protein expression test
  • the eGFP coding sequence was used as a DNA template, and the cap analogues of Examples 1-4 and Comparative Examples 1-2 were used as a starting point for in vitro transcription. The different mRNA products obtained were then transfected into 293T cells.
  • 293T cells are plated at (0.5-1) ⁇ 10 5 cells (24-well plate). It is recommended to use cells within 50 generations for transfection experiments. It is required to re-passage the cells 24 hours before transfection in culture medium. Adding antibiotics has no effect on transfection efficiency. During transfection, the cell density is generally 60-80%. Each well is transfected with 2 ⁇ g of mRNA. Use Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) as the transfection reagent and refer to its usage instructions for operation. The transfected cells were placed in a 37°C, CO 2 incubator and replaced with fresh complete culture medium 4-6 hours after transfection.

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Abstract

本发明提供了一种含开环核苷结构的起始加帽寡核苷酸引物，该含开环核苷结构的起始加帽寡核苷酸引物的分子结构式为m7UNGpppA2'OmepG，本发明提供的含开环核苷结构的起始加帽寡核苷酸引物具有更高的mRNA体外转录效率、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

一种含开环核苷结构的起始加帽寡核苷酸引物
本申请要求于2022年05月05日提交中国专利局、申请号为202210480431.7、发明名称为“一种含开环核苷结构的起始加帽寡核苷酸引物”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
本发明涉及化学及生物工程技术领域，具体涉及一种含开环核苷结构的起始加帽寡核苷酸引物。
背景技术
在真核细胞中，大多数信使RNA(mRNA)的5'末端被封闭，或“帽化(加帽)”，所述帽包含有在两个核苷部分之间的5'-5'三磷酸键合和远端鸟嘌呤环上的7-甲基，mRNA的帽化促进其在细胞中的正常功能。通过体外转录合成mRNA已经成为引入外源基因并进行表达蛋白的重要工具，并广泛应用于疾病的治疗和预防中，体外转录合成mRNA使得工作人员能够制备在各种生物学应用中表现适当的RNA分子。此类应用包括多肽的研究应用和商业生产，例如，在无细胞翻译体系中产生在特定位点包含“非天然”氨基酸的多肽，或在培养的细胞中产生就其活性或稳定性而言需要翻译后修饰的多肽。在后者体系中，合成进行显著更长的时间，并因此产生更多的蛋白质。mRNA的体外转录产率以及5’位加帽类似物是mRNA制备过程的关键工艺。目前应用于mRNA化学法加帽的体系，无法获得较高的效率。
专利CN201680067458.6报道了用于合成5’-加帽RNA的组合物和方法。其中起始加帽寡核苷酸引物具有通式形式m7Gppp[N2，Ome]n[N]m，其中m7G为N7-甲基化的鸟苷或任何鸟苷类似物，N为任何天然的、修饰的或非天然的核苷，“n”可以是从0至4的任何整数且“m”可以是从1至9的整数。Cleancap属于Cap1，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。
专利US10968248B2公开了Trinucleotide mRNA cap analogs，涉及三核苷酸帽类似物，用于改善体外mRNA合成和m7G(5’)p3-RNA转录。该结构中 在帽子结构中的第三个核苷酸采用开环的UNA替代，由于是转录的起始核苷酸，不利于T7RNA聚合酶的识别，降低了加帽效率，也降低了体外转录的效率。
因此，本领域迫切需要开发一类新的加帽类似物组合，从而使体外转录合成的mRNA能够获得更高的体外转录产量、更高的加帽效率、更低的免疫原性。
发明内容
为了解决现有技术中体外转录产量以及加帽效率不足等问题。本申请提供了一种含开环核苷结构的起始加帽寡核苷酸引物，该含开环核苷结构的起始加帽寡核苷酸引物结构中含有UNA结构替换原有的五元糖环结构，替换后由于UNA无法作为转录的起始位点，具有很好的mRNA体外转录时抗反转录效果，最终使mRNA获得更高的加帽效率；UNA的开环结构，帮助mRNA逃脱体内免疫系统识别，更好的降低免疫原性；同时，由于非天然核苷酸UNA的引入，使得mRNA不容易被核酶水解，增加了mRNA加帽后在体内的稳定性。
一种含开环核苷结构的起始加帽寡核苷酸引物，其包含以下结构：
其中，R1和R2独立的为H、OH、烷基、O-烷基、卤素；
X1、X2和X3分别独立的为O、CH2或NH；
Y1、Y2和Y3分别独立的为O、S、Se或BH3；
Ra和Rb独立的为
R3和R4独立的为氢、羟基、取代或未取代的O-烷基、取代或未取代的S-烷基、取代或未取代的NH-烷基、取代或未取代的N-二烃基、取代或未取代的O-芳香基、取代或未取代的S-芳香基、取代或未取代的NH-芳香基、取代或未取代的O-芳烷基、取代或未取代的S-芳烷基、取代或未取代的NH-芳烷基；
B1和B2独立的为天然的、或修饰的、或非天然的核苷碱基。
该含开环核苷结构的起始加帽寡核苷酸引物的制备方法，包括以下步骤：(1)m7UrGDP-Im的合成：从鸟苷出发合成糖环开环核苷，在开环核苷的基础上依次进行二磷酸化、N7的甲基化、多磷酸的咪唑化反应，合成m7UrGDP-Im；(2)磷酸酯键连接的二核苷酸的制备：通过开环或非开环的亚磷酰胺单体与开环或非开环的双取代核苷单体，在四氮唑的作用下偶联形成第一个磷酸酯键，通过酸作用，脱除保护基，然后引入第二个磷酸，最终水解得到磷酸酯键连接的二核苷酸；(3)含开环核苷结构的起始加帽寡核苷酸引物的合成：m7UrGDP-Im与磷酸酯键连接的二核苷酸反应制备含开环核苷结构的起始加帽寡核苷酸引物；
上述亚磷酰胺单体结构式为：
其中，R5和R6独立的为H、OH、烷基、O-烷基、卤素；B3和B4独立的为天然的、或修饰的、或非天然的核苷碱基。
上述双取代核苷单体选自中的任一种。
该含开环核苷结构的起始加帽寡核苷酸引物的制备方法，具体包括以下步骤：
(1)m7UrGDP-Im的合成：
(1-1)称取鸟苷，分散在DMF中，冰浴使反应液内温控制在5～10℃，分两个批次加入TBSCl；反应结束后加入水将产物析出，过滤并洗涤滤饼得目 标化合物B；
(1-2)称取化合物B分散在乙腈中，加入高碘酸钠，加热反应到50±5℃；反应结束后加入水中过滤得目标化合物C；
(1-3)称取化合物C溶解在无水甲醇，反应液冷却至0±5℃，加入硼氢化钠，充分搅拌，HPLC监控反应，反应结束后缓慢加入冰水，猝灭结束后浓缩干得化合物D，再将加合物溶D解在水中，用2M的盐酸调节PH到3，反向色谱纯化，得目标化合物E；
(1-4)将化合物E溶解在磷酸三甲酯中，反应液冷却至0±5℃，缓慢滴加三氯氧磷，低温反应4～5小时后，加入2M的醋酸铵溶液猝灭反应，反相色谱纯化得目标化合物F，得到的化合物F与三苯基膦、二硫二吡啶、咪唑充分反应，反应液加入4M的高氯酸钠丙酮溶液中析出，滤饼用丙酮充分洗涤得目标化合物G；
(1-5)称取目标化合物G溶解DMF中，加入磷酸三丁胺，充分搅拌得目标化合物H，向反应液中加入水溶液，反应液冷却至0±5℃，缓慢滴加硫酸二甲酯，过程中用2M的氢氧化钠调节PH不超过5，HPLC监测反应，反应结束后离子色谱纯化得目标化合物I；
(1-6)将化合物I溶解在DMF中，与三苯基膦、二硫二吡啶、咪唑充分反应，反应液加入4M的高氯酸钠丙酮溶液中析出，滤饼用丙酮充分洗涤得目标化合物m7UrGDP-Im；
(2)磷酸酯键连接的二核苷酸的制备：
称取开环或非开环亚磷酰胺单体于单口瓶中，用二氯甲烷溶解，再加入开环或非开环双取代核苷单体，降温至25±2℃，氮气鼓吹下加入四氮唑，升温至25±2℃反应；监测反应结束后，将碘吡啶溶液加入到反应液中，监测反应结束后旋干，浓缩后的油膏溶解在二氯甲烷中，加入三氟乙酸；监测反应结束后，旋干，石油醚/二氯甲烷按一定比例打浆，过滤得中间体A2；将A2溶解在乙腈中，加入膦试剂、四氮唑充分搅拌反应；监测反应结束后，再将碘吡啶溶液加入到反应液中，监测反应结束后旋干，在旋瓶中加入甲醇和浓氨水，室温反应4小时，监测反应；反应结束后旋干，加入超纯水，进入反向离子渗透设备，洗涤浓缩，冻干得目标化合物磷酸酯键连接的二核苷酸；
(3)含开环核苷结构的起始加帽寡核苷酸引物的合成：
将步骤(1)得到的m7UrGDP-Im溶解在含有MnCl2的DMF溶液中，并添加到步骤(2)得到的磷酸酯键连接的二核苷酸的DMF溶液中，在室温下搅拌反应，24小时后，用0.25M EDTA溶液终止反应；将混合物装载到DEAESephadex柱(30×500cm)上。将产物使用0-1.0M的TEAB洗脱液线性梯度洗脱。收集HPLC纯度＞97％的洗脱产物，浓缩完以上分离液，再装载到强阴离子树脂，使用0-1.0M的醋酸钠洗脱液线性梯度洗脱，收集HPLC纯度＞98.5％的洗脱产物，合并高纯度洗脱液，通过纳滤设备去除残留的醋酸钠溶液并浓缩得目标产物含开环核苷结构的起始加帽寡核苷酸引物。
本发明提供了一种含开环核苷结构的起始加帽寡核苷酸引物，该含开环核苷结构的起始加帽寡核苷酸引物的分子结构式为m7UNGpppA2’OmepG，本发明提供的含开环核苷结构的起始加帽寡核苷酸引物适用于以DNA序列为模板利用体外共转录方法生产的mRNA，该DNA序列可以来源或改造自病毒、动物、植物等物种，同时其生产的mRNA具有更高的体外转录效率、更高的加帽效率、更低的免疫原性和更高的蛋白翻译效率。
本发明相比现有技术具有以下优点：
与现有帽结构类似物Cleancap相比，本发明的含开环核苷结构的起始加帽寡核苷酸引物具有更高的合成效率、更高的加帽效率、更低的免疫原性、更高的蛋白翻译效率。
附图说明
图1是实施例1的含开环核苷结构的起始加帽寡核苷酸引物起始的mRNA转录的加帽率检测结果图；
图2是实施例2的含开环核苷结构的起始加帽寡核苷酸引物起始的mRNA转录的加帽率检测结果图；
图3是实施例3的含开环核苷结构的起始加帽寡核苷酸引物起始的mRNA转录的加帽率检测结果图；
图4是实施例4的含开环核苷结构的起始加帽寡核苷酸引物起始的mRNA转录的加帽率检测结果图；
图5是对比例1的帽类似物起始的mRNA转录的加帽率检测结果图；
图6是对比例2的帽类似物起始的mRNA转录的加帽率检测结果图；
图7是实施例1-4及对比例1-2的细胞表型图；
图8是实施例1-4及对比例1-2的荧光统计结果图。
具体实施方式
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整的描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
各实施例中所使用的原料名称及来源参见下表1：
表1


以下各实施例中所使用的m7UrGDP-Im(J)均通过以下步骤制备得到：
(1)称取5g鸟苷，分散在50mL的DMF中，冰浴使反应液内温低于10℃，分两个批次加入1.2eq的TBSCl，HPLC监控反应至原料≤5％，反应结束后加入100mL的水将产物析出，过滤并洗涤滤饼得目标化合物B；
(2)称取2g化合物B分散在20mL乙腈中，加入1.2eq的高碘酸钠，加热反应到50℃，12h后监测反应，反应结束后加入40mL水中过滤得目标化合物C；
(3)称取2g化合物C溶解在无水甲醇，反应液冷却至4℃，加入5eq得硼氢化钠，充分搅拌，HPLC监控反应，反应结束后缓慢加入冰水，猝灭结束后浓缩干得化合物D，再将加合物溶D解在水中，用2M的盐酸调节PH到3，反向色谱纯化，得目标化合物E；
(4)将2g化合物E溶解在10ml的磷酸三甲酯中，反应液冷却至0℃，缓慢滴加1.2eq三氯氧磷，低温反应4小时后，加入2M的醋酸铵溶液猝灭反 应，反相色谱纯化得目标化合物F，得到的化合物F与1eq的三苯基膦，2eq的二硫二吡啶，4eq的咪唑充分反应，反应液加入4M的高氯酸钠丙酮溶液中析出，滤饼用丙酮充分洗涤得目标化合物G；
(5)称取2g目标化合物G溶解DMF中，加入3eq的磷酸三丁胺，充分搅拌得目标化合物H，向反应液中加入20eq的水溶液，反应液冷却至4℃，缓慢滴加硫酸二甲酯，过程中用2M的氢氧化钠调节PH不超过5，HPLC监测反应，反应结束后离子色谱纯化得目标化合物I；
(6)将4g化合物I溶解在50mL DMF中，与1eq的三苯基膦，2eq的二硫二吡啶，4eq的咪唑充分反应，反应液加入4M的高氯酸钠丙酮溶液中析出，滤饼用丙酮充分洗涤得目标化合物J；
m7UrGDP-Im(J)具体的反应路线流程，如下方程式(1)：
合成例1中所使用A-G-P的合成路线为：称取5kg2’OMe-rA亚磷酰胺单体于单口瓶中，用50L的二氯甲烷溶解，再加入2.73kg2’,3’乙酰基鸟苷，降温至25±2℃，氮气鼓吹下加入880g四氮唑，升温至25±2℃反应。监测反应结束后，将1.2eq的碘吡啶溶液加入到反应液中，监测反应结束后旋干，浓缩后的油膏溶解在4L二氯甲烷中，加入1.1eq的三氟乙酸，监测反应结束后，旋干，石油醚/二氯甲烷按一定比例打浆，过滤得中间体A2；将A2溶解在4L乙腈中，加入1.2eq的膦试剂、1.2eq的四氮唑充分搅拌反应，监测反应结束后，再加入1.2eq的碘吡啶溶液加入到反应液中，监测反应结束后旋干，在旋瓶中加入3L甲醇和3L浓氨水，室温反应4小时，监测反应，反应结束后旋干，加入20L超纯水，进入反向离子渗透设备，洗涤浓缩，冻干得目标化合物A-G-P，反应路线流程，如下方程式(2)：
合成例2中所使用的A-UrG-P的合成方法参考实施例1中A-G-P合成方法，A-UrG-P的反应路线流程，如下方程式(3)：
其中D的合成参考中间体J的合成步骤；E2的制备包括以下步骤：称取20g化合物D溶解在乙腈中，加入3eq的三乙胺，反应液冷却至4℃，缓慢滴加乙酸酐，反应结束后，加入2eq的TBAF，脱除TBS保护基旋干柱层析得化合物E2；化合物E2取代双取代鸟苷得到A-UrG-P。
合成例3中所使用的UrA-G-P的合成方法参考实施例1中A-G-P合成方法，UrA-G-P的反应路线流程，如下方程式(4)，
其中D的合成参考中间体J的合成步骤；F4的制备包括以下步骤：(1)称取10g化合物D溶解在DMF中，冰浴，缓慢加入1.2eq的NaH，低温搅拌 2h后，缓慢滴加碘甲烷2eq，室温反应三小时后，加水猝灭反应，过滤得到化合物F1的粗品，反向色谱纯化；(2)称取2g化合物F分散在30mL的甲醇中，加入2eq的TBAF，2小时后反应结束，旋干直接下一步反应；将旋干的固体溶解在30ml的DCM中，加入1.2eq的三乙胺，冰浴搅拌20min，缓慢加入DMTr-Cl的DCM溶液，滴加结束后反应半小时，柱层析得目标化合物F3；(3)称取3g化合物F3，通过2eq的TMSCl转移保护，对6’位置的胺基Bz化转移保护，纯化后通过与四氮唑和膦试剂反应得到目标化合物F4；化合物F4取代2’OMe-rA亚磷酰胺单体得到UrA-G-P。
合成例4中所使用的UrA-UrG-P的合成方法参考实施例1中A-G-P合成方法，UrA-UrG-P通过E2和F4反应得到，反应路线流程，如下方程式(5)：
实施例1：Ra和Rb均为五元糖环的含开环核苷结构的起始加帽寡核苷酸引物的合成方法
以m7UrGDP-Im(J)和A-G-P为原料通过以下步骤合成：将m7UrGDP-Im(J)(2mol)溶解在含有MnCl2(0.2mol)的DMF溶液中，并添加到A-G-P(1.8mol)的DMF溶液中。在室温下搅拌反应。24小时后，用10L的0.25M EDTA溶液终止反应。将混合物装载到DEAESephadex柱(30×500cm)上。将产物使用0-1.0M的TEAB洗脱液线性梯度洗脱。收集HPLC纯度＞97％的洗脱产物，浓缩完以上分离液，再装载到强阴离子树脂，使用0-1.0M的醋酸钠洗脱液线性梯度洗脱，收集HPLC纯度＞98.5％的洗脱产物，合并高纯度洗脱液，通过纳滤设备去除残留的醋酸钠溶液并浓缩得目标产物，反应路线流程，如下方程式(6)：
实施例2：Ra为五元糖环、Rb为开环结构的含开环核苷结构的起始加帽寡核苷酸引物
本实施例含开环核苷结构的起始加帽寡核苷酸引物以m7UrGDP-Im(J)和A-UrG-P为原料，参考实施例1目标产物的合成方法得到。
实施例3：Ra为开环结构、Rb为五元糖环的含开环核苷结构的起始加帽寡核苷酸引物
本实施例含开环核苷结构的起始加帽寡核苷酸引物以m7UrGDP-Im(J)和UrA-G-P为原料，参考实施例1目标产物的合成方法得到。
实施例4：Ra、Rb均为开环结构的含开环核苷结构的起始加帽寡核苷酸引物
本实施例含开环核苷结构的起始加帽寡核苷酸引物以m7UrGDP-Im(J)和UrA-UrG-P为原料，参考实施例1目标产物的合成方法得到。
对比例1：m7GpppA2’OmepG
m7GpppA2’OmepG的合成方法参考上述实施例的合成方法，反应路线流程，如下方程式(7)：
对比例2：仅有Rb为开环结构的加帽类似物，合成方法参考上述实施例的合成方法，反应路线流程，如下方程式(8)：
各实施例所得到的含开环核苷结构的起始加帽寡核苷酸引物以及对比例所得到的加帽类似物结构如下表2所示，
表2

测试例1：mRNA体外转录产量及加帽效率的测定
利用含开环核苷结构的起始加帽寡核苷酸引物进行mRNA的体外合成：先用NotI线性化质粒，4℃酶切过夜；DNA模板抽提；体外转录合成mRNA，分别使用实施例1-4含开环核苷结构的起始加帽寡核苷酸引物及对比例1-2的加帽类似物作为帽结构。
反应体系如表3：
表3

备注：在实验过程中，首先计算好体系所需物料体积，然后进行加样。首先在体系中加入无菌无酶水，随后依次加入10X buffer、NTPs、帽结构，混匀后轻轻离心，随后加入核酸酶抑制剂、无机焦磷酸酶、T7RNA聚合酶、线性化DNA模板，充分混匀后轻轻离心，于37℃下孵育。2小时后加入DNase I 1U，37℃继续孵育30分钟以去除DNA模板，然后进行RNA纯化，通常使用磁珠纯化方法。纯化的mRNA用无菌无酶水进行溶解，随后利用Nanodrop One进行定量检测。
液相色谱质谱法(LC-MS)被用来检测不同起始帽类似物的mRNA的IVT加帽率；首先需要设计一段与转录产物mRNA起始碱基匹配的具有标记的DNA探针，通常的标记为biotin标记，将链霉亲和素标记的磁珠清洗后与合成的DNA探针、mRNA及10×RNase H reaction buffer室温室温孵育30分钟，边孵育边缓慢混匀，随后加入20ul RNase H(5U/ul)孵育37度3h，每半个小时混匀一次。孵育结束后对磁珠进行清洗，清洗完成后的磁珠加入100μL加热到80℃的75％甲醇，混合物在加热板上加热到80℃，保持3分钟，然后放置磁力架上吸取上清，使用蒸发离心机在室温下干燥45分钟至10μl。然后将样品重新悬浮在50μl的100μM EDTA/1％MeOH中，即可用于LC-MS分析，确定转录反应中RNA的加帽情况。由于加帽与非加帽的碱基在分子量上有明显区别，利用分子质量差别即可判定不同帽类似物起始的mRNA转录的加帽率。具体结果见表4及说明书附图1-6：图1-6，横坐标表示分子量，不同分子量与不同结构一一对应，通过分子量我们可以推测出所含的碱基及其修饰，单个峰下的峰面积为该分子量大小下对应的比例，通过统计不同分子量下的峰面积 进而可以推算出不同修饰和帽结构在产物中的比例；其中图1-3测试样本模板使用的模板酶切后的序列为m7G-pppAGGCGCCACCAUGGUGAGCA(所得到的分子量在6977左右)，图4-6测试样本使用的模板酶切后的序列为m7G-pppGGGCGCCACCAUGGUGAGCAA(所得到的分子量在7322左右)。
表4
以上表4中数据是实例1-4以及对比例1-2在相同的实验条件下的mRNA体外转录产量以及加帽效率结果。我们发现对比例1的产量50ug，实施例1的产量提高至72ug，提高了44％，并且对比例1的加帽率为93.3％，实施例1的加帽率提高至99.3％，提高了6％。因此，由实验结果可知，本申请的含开环核苷结构的起始加帽寡核苷酸引物具有较高的mRNA体外转录产量以及加帽效率。
测试例2：mRNA与RIG I结合能力的测定
RIG-I主要包括N端两个重复的caspase活化和募集结构域(caspase activation and recruitment domain,CARD)，位于中间的解螺旋酶结构和C端RNA结构域。RIG-I的N端CARD结构域即使在没有病毒感染的条件下，过表达结构域也能够促进细胞分泌I型干扰素(IFN)，因此，该结构域主要负责向下游传递信号。
本研究将利用实施例1-4含开环核苷结构的起始加帽寡核苷酸引物及对比例1-2的帽类似物为起始进行体外转录的eGFP的mRNA转染293T细胞，24小时后收集细胞，利用RNA免疫共沉淀的方法将胞内蛋白RIG-I与其结合的RNA一起进行免疫共沉淀，最后对这些mRNA进行逆转录以及实时定量PCR。
具体的细胞的培养条件同上，转染24h后收集细胞，首先加入固定液孵育，10min后加入合适浓度的甘氨酸溶液终止反应，收集细胞。利用裂解液对收集的细胞进行裂解，12000rpm，4℃离心10min，取上清分别与的RIG-I或IgG抗体，4度摇床孵育过夜。随后向其中加入20μl Protein A/G磁珠，4℃孵育4h后，在磁力架上进行洗涤，洗涤完成后，提取RNA即可用于后续RT-qPCR验证表达结果。不同帽类似物核苷酸mRNA与RIG-1的结合能力结果如下表5：
表5
由上表5的实验数据可知，本发明中的含开环核苷结构的加帽类似物应用于mRNA合成细胞免疫原性明显低于Cleancap。
测试例3：细胞蛋白表达测试
采用eGFP编码序列为DNA模板，利用实施例1-4及对比例1-2的帽类似物为起始进行体外转录。随后将获得的不同的mRNA产物进行293T细胞的转染。
293T细胞以(0.5-1)×105个细胞进行铺板(24孔板)，推荐使用在50代以内的细胞进行转染实验。要求在转染前24小时对细胞再次传代，在培养基中 加入抗生素对转染效果没有影响。转染时细胞密度一般60-80％为佳，每孔转染2μg mRNA，转染试剂选用Lipofectamine MessengerMAX Transfection Reagent(Invitrogen)并参考其使用方法进行操作。转染后的细胞放置在37℃，CO2孵育箱中，转染4-6小时后，更换为新鲜的完全培养基。在37℃的CO2培养箱箱中孵育24小时以后，荧光显微镜观察其中GFP的荧光强度。结果见图7和图8，结果中可以明显的看到本发明的mRNA的表达效率要明显高于对比例，同时两者均未引起明显的细胞死亡，这一结果表明本申请的含开环核苷结构的起始加帽寡核苷酸引物具有更高的表达效率；即本发明中的含开环核苷结构的起始加帽寡核苷酸引物应用于mRNA合成有效蛋白翻译效率明显高于Cleancap(对比例1)帽结构的蛋白翻译效率。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (2)

  1. 一种含开环核苷结构的起始加帽寡核苷酸引物，其特征在于，其包含以下结构：
    其中，R1和R2独立的为H、OH、烷基、O-烷基、卤素；
    X1、X2和X3分别独立的为O、CH2或NH；
    Y1、Y2和Y3分别独立的为O、S、Se或BH3；
    Ra和Rb独立的为
    R3和R4独立的为氢、羟基、取代或未取代的O-烷基、取代或未取代的S-烷基、取代或未取代的NH-烷基、取代或未取代的N-二烃基、取代或未取代的O-芳香基、取代或未取代的S-芳香基、取代或未取代的NH-芳香基、取代或未取代的O-芳烷基、取代或未取代的S-芳烷基、取代或未取代的NH-芳烷基；
    B1和B2独立的为天然的、或修饰的、或非天然的核苷碱基。
  2. 权利要求1所述的含开环核苷结构的起始加帽寡核苷酸引物的制备方法，其特征在于，包括以下步骤：(1)m7UrGDP-Im的合成：从鸟苷出发合成糖环开环核苷，在开环核苷的基础上依次进行二磷酸化、N7的甲基化、多磷酸的咪唑化反应，合成m7UrGDP-Im；(2)磷酸酯键连接的二核苷酸的制备：通过开环或非开环的亚磷酰胺单体与开环或非开环的双取代核苷单体，在四氮唑的作用下偶联形成第一个磷酸酯键，通过酸作用，脱除保护基，然后引入第二个磷酸，最终水解得到磷酸酯键连接的二核苷酸；(3)含开环核苷结构的起始加帽寡核苷酸引物的合成：m7UrGDP-Im与磷酸酯键连接的二核苷酸反应制备含开环核苷结构的起始加帽寡核苷酸引物；
    所述亚磷酰胺单体结构式为：
    其中，R5和R6独立的为H、OH、烷基、O-烷基、卤素；B3和B4独立的为天然的、或修饰的、或非天然的核苷碱基。
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