CN115057903A - Initial capped oligonucleotide primer containing morpholine ring structure and preparation method and application thereof - Google Patents

Initial capped oligonucleotide primer containing morpholine ring structure and preparation method and application thereof Download PDF

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CN115057903A
CN115057903A CN202210716120.6A CN202210716120A CN115057903A CN 115057903 A CN115057903 A CN 115057903A CN 202210716120 A CN202210716120 A CN 202210716120A CN 115057903 A CN115057903 A CN 115057903A
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缪佳颖
黄磊
赵万年
沈奇
肖潇
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Jiangsu Shenji Biotechnology Co ltd
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Abstract

The invention provides an initial capped oligonucleotide primer containing a morpholine ring structure, and a preparation method and application thereof, wherein the molecular formula of the initial capped oligonucleotide primer containing the morpholine ring structure is m7 GpppA 2'OMe pG, wherein m7 G is N 7 -methylated guanosine or any guanosine analogue, a being any natural, modified or non-natural adenosine or any adenosine analogue. The mRNA synthesized by the initial capped oligonucleotide primer containing the morpholine ring structure has lower immunogenicity, higher protein translation efficiency and higher stability.

Description

Initial capped oligonucleotide primer containing morpholine ring structure and preparation method and application thereof
Technical Field
The invention relates to the technical field of chemical and biological engineering, in particular to an initial capping oligonucleotide primer containing a morpholine ring structure, and a preparation method and application thereof.
Background
In eukaryotic cells, the 5' end of most messenger rnas (mrnas) is blocked, or "capped," which contains a 5' -5' triphosphate linkage between two nucleoside moieties and a 7-methyl group on the distal guanine ring, capping of the mRNA facilitates its normal function in the cell. The synthesis of mRNA by in vitro transcription has become an important tool for the introduction of foreign genes and the expression of proteins, and is widely used in the treatment and prevention of diseases, enabling workers to prepare RNA molecules that exhibit the right expression in various biological applications. Such applications include research applications and commercial production of polypeptides, for example, production of polypeptides comprising "unnatural" amino acids at specific sites in cell-free translation systems, or production of polypeptides in culture that require post-translational modification for their activity or stability. In the latter system, synthesis takes significantly longer and therefore more protein is produced.
Patent CN201680067458.6 reports compositions and methods for synthesizing 5' -capped RNA. Wherein the initial capped oligonucleotide primer has the general form m 7 Gppp[N 2’OMe ] n [N] m Wherein m is 7 G is N 7 -methylated guanosine or any guanosine analogue, N being 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, and uses dimer (m) with ARCA 7 GpppG) initiates transcription of T7 differently, CleanCap uses trimer (m) 7 GpppAmG) initiates transcription of T7. The method has high yield, and can prepare 4mg of capped RNA per ml of transcription reaction system, with capping efficiency up to 90%, and immunogenicity of transcription productLower than ARCA.
The 5' capping analog is a key structure for reducing the immunogenicity of mRNA, and the immunogenicity of mRNA can be reduced by optimizing the structure of the capping analog. Therefore, there is a need to continually optimize the structure of cap analogs to find new cap analogs that are less immunogenic than the currently available clearcap. According to the invention, the morpholine ring structure is used for replacing the pentasaccharide ring in the structure of the cap analogue, so that the low binding capacity of mRNA and RIG-I can be reduced, and the immunogenicity of the mRNA can be obviously reduced.
Disclosure of Invention
In order to further improve the stability of RNA, prolong the half-life of a medicament, obviously improve the stability of mRNA and reduce the low binding capacity of mRNA and RIG-I, the application provides an initial capping oligonucleotide primer containing a morpholine ring structure, a preparation method and application thereof. Meanwhile, mRNA synthesized by the cap analogue with the morpholine ring structure has lower binding capacity with RIG-I, and shows lower immunogenicity.
An initial capped oligonucleotide primer comprising a morpholino ring structure comprising the structure:
Figure BDA0003707070850000021
wherein, X 1 、X 2 And X 3 Are each independently O, CH 2 Or NH;
Y 1 、Y 2 and Y 3 Each independently is O, S, Se or BH 3 ；
R a Is composed of
Figure BDA0003707070850000022
Rb is
Figure BDA0003707070850000023
And when Ra is
Figure BDA0003707070850000024
When Rb is
Figure BDA0003707070850000025
R 1 Is hydrogen, hydroxy, substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted N-dihydrocarbyl, 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;
R 2 and R 3 Independently H, OH, alkyl, O-alkyl, halogen;
B 1 and B 2 Independently, a natural, or modified, or non-natural nucleobase.
The preparation method of the initial capped oligonucleotide primer containing the morpholine ring structure comprises the following steps: (1) synthesis of intermediate F: synthesizing a compound A from guanosine, and successively carrying out diphosphorylation, methylation of N7 and imidization of polyphosphoric acid on the basis of the compound A to synthesize an intermediate F; (2) preparation of a phosphate-linked dinucleotide: coupling a phosphoramidite monomer and a disubstituted nucleoside monomer under the action of tetrazole to form a first phosphate ester bond, removing a protecting group through acid action, introducing a second phosphoric acid, and finally hydrolyzing to obtain a dinucleotide connected with the phosphate ester bond; (3) synthesis of initial capped oligonucleotide primers containing a morpholine ring structure: reacting the intermediate F with dinucleotide connected with a phosphate bond to prepare an initial capped oligonucleotide primer containing a morpholine ring structure;
the phosphoramidite monomer has a structural formula:
Figure BDA0003707070850000031
wherein R4 is H, OH, alkyl, O-alkyl, halogen;B 3 and B 4 Independently, a natural, or modified, or non-natural nucleobase.
The above disubstituted nucleoside monomer is selected from
Figure BDA0003707070850000032
Any one of the above.
The preparation method of the initial capped oligonucleotide primer containing the morpholine ring structure specifically comprises the following steps:
(1) synthesis of intermediate F:
(1-1) suspending guanosine in ethanol and adding NaIO 4 Dissolved in water at 40 + -5 deg.C and added dropwise to the adenosine suspension with vigorous stirring. After 15min, (NH) was added 4 ) 2 B 4 O 7 .4H 2 And O. And (3) dropwise adding triethylamine to keep the pH value of a reaction system between 8.5 and 9.0, reacting at room temperature for 1.5 hours, and monitoring the reaction by HPLC (high performance liquid chromatography) until adenosine is less than or equal to 1%. Suction filtration and washing of the filter cake with ethanol. Adding NaBH to the filtrate in batches under ice bath 3 CN was stirred at room temperature for 1 hour, the pH was adjusted to 3-4 with TFA, the reaction was continued for 2 hours, and the completion of the reaction was monitored by HPCL. The residual TFA was distilled off under reduced pressure. The crude product is prepared, purified and freeze-dried in reverse phase to obtain a compound A;
(1-2) dissolving the compound A in trimethyl phosphate, cooling the reaction liquid to 0 +/-5 ℃, slowly dropwise adding phosphorus oxychloride, reacting at room temperature for 4 hours, adding 2M ammonium acetate solution for quenching reaction, preparing and purifying in a reversed phase manner, and freeze-drying to obtain a target compound B;
(1-3) suspending the compound B with triphenylphosphine, dithiodipyridine, imidazole and TEA in DMF, reacting at room temperature for 8h, monitoring the reaction by HPLC until the raw material is less than or equal to 1%, adding 4M sodium perchlorate acetone solution into the reaction solution, performing suction filtration, and fully washing the filter cake with acetone to obtain a target compound C;
(1-4) weighing a target compound C, suspending in DMF, adding triethylamine phosphate and manganese chloride, stirring at room temperature for 8 hours, monitoring the reaction by HPLC (high performance liquid chromatography) until the raw material is less than or equal to 1%, and pouring the reaction liquid into water to obtain a crude product water solution of the compound D. Slowly dropwise adding dimethyl sulfate, adjusting the pH value to be not more than 5 by using 2M sodium hydroxide in the process, monitoring the reaction by using HPLC, and after the reaction is finished, purifying by using ion chromatography and freeze-drying to obtain a target compound E;
(1-5) suspending the compound E and triphenylphosphine, dithiodipyridine, imidazole and TEA in DMF, reacting for 8h at room temperature, monitoring the reaction by HPLC until the raw materials are less than or equal to 1%, adding 4M sodium perchlorate acetone solution into the reaction solution, performing suction filtration, and fully washing the filter cake with acetone to obtain a target compound F;
(2) preparation of a phosphate-linked dinucleotide:
weighing 2 ' OMe-rA phosphoramidite monomer, dissolving in dichloromethane, adding 2 ', 3 ' acetyl guanosine, cooling to 25 +/-2 ℃, adding tetrazole under nitrogen blowing, and heating to 25 +/-2 ℃ for reaction. After the monitoring reaction is finished, adding an iodopyridine solution into the reaction solution, performing spin drying after the monitoring reaction is finished, dissolving the concentrated ointment into dichloromethane, adding trifluoroacetic acid, performing spin drying after the monitoring reaction is finished, pulping petroleum ether/dichloromethane according to a certain proportion, and filtering to obtain an intermediate G1; dissolving G1 in acetonitrile, adding a phosphine reagent and tetrazole, fully stirring for reaction, after monitoring reaction is finished, adding an iodopyridine solution into a reaction solution, after monitoring reaction is finished, carrying out spin-drying, adding methanol and concentrated ammonia water into a spinner bottle, reacting at room temperature for 4 hours, monitoring reaction, after reaction is finished, carrying out spin-drying, adding ultrapure water, entering a reverse ion permeation device, washing, concentrating, and freeze-drying to obtain the dinucleotide connected with the phosphate bond;
(3) synthesis of initial capped oligonucleotide primers containing a morpholine ring structure:
dissolving m7UrGDP-Im in MnCl 2 Adding the intermediate F obtained in step (1) to the DMF solution containing the phosphate-linked dinucleotide, and dissolving the intermediate F in the solution containing MnCl 2 And adding the solution into the DMF solution of A-G-P obtained in the step (2). The reaction was stirred at room temperature. After 24h, the reaction was stopped with 0.25M EDTA solution. The mixture was loaded onto a DEAE Sephadex column (30X 500 cm). The product was eluted using a linear gradient of 0-1.0M TEAB eluent. Collecting the eluate with HPLC purity of more than 97%, concentrating the above separated solution, loading to strong anion resin, eluting with 0-1.0M sodium acetate eluate in linear gradient, collecting the eluate with HPLC purity of more than 98.5%,and combining the high-purity eluents, removing residual sodium acetate solution by nanofiltration equipment, and concentrating to obtain the target product of the initial capped oligonucleotide primer containing the morpholine ring structure.
The initial capping oligonucleotide primer containing the morpholine ring structure is used for mRNA capping under a T7 RNase system. T7 RNA polymerase is a DNA-dependent RNA polymerase with high specificity for the bacteriophage T7 promoter sequence. This enzyme synthesizes large amounts of RNA from the insertion of the T7 promoter into DNA downstream of the transcription vector. The IVT (in vitro transcription) reaction system catalyzed by T7 RNA polymerase is the most mature mRNA preparation system at present.
The invention provides an initial capping oligonucleotide primer containing a morpholine ring structure, which is suitable for mRNA produced by using a DNA sequence as a template by an in vitro co-transcription method, wherein the DNA sequence can be derived or modified from virus, animals, plants and other species, and the produced mRNA has lower immunogenicity, higher protein translation efficiency and better stability.
Compared with the prior art, the invention has the following advantages:
compared with the existing cap structure analog Cleancap, the mRNA produced by the initial capping oligonucleotide primer containing the morpholine ring structure has lower immunogenicity, higher protein translation efficiency and better stability.
Drawings
FIG. 1 is a cell phenotype diagram of examples 1 to 3 and comparative examples 1 to 2;
FIG. 2 is a graph showing fluorescence statistics of examples 1 to 3 and comparative examples 1 to 2;
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The raw material names and sources used in each example are given in table 1 below:
TABLE 1
Figure BDA0003707070850000061
Figure BDA0003707070850000071
The intermediate F used in each of the following examples was prepared by the following procedure:
(1-1) suspending 5.0g of guanosine in 100mL of ethanol, and adding 1.2eq. NaIO 4 Dissolved in 150mL of warm water at 40 ℃ and added dropwise to the adenosine suspension with vigorous stirring. After 15min, (NH) was added 4 ) 2 B 4 O 7 .4H 2 O (1.2 eq.). Triethylamine (about 5.0mL) was added dropwise to maintain the pH of the reaction system at 8.5 to 9.0, and after 1.5 hours of reaction at room temperature, the reaction was monitored by HPLC until adenosine became less than or equal to 1%. Suction filtration and washing of the filter cake with ethanol. Adding NaBH to the filtrate in batches under ice bath 3 CN (1.3eq.), stirring at room temperature for 1h, adjusting pH to 3-4 with TFA, continuing the reaction for 2h, and monitoring the completion of the reaction with HPCL. The residual TFA was distilled off under reduced pressure. The crude product is prepared, purified and freeze-dried in reverse phase to obtain a compound A;
(1-2) dissolving 2g of the compound A in 20ml of trimethyl phosphate, cooling the reaction liquid to 0 ℃, slowly dropwise adding 2.0eq of phosphorus oxychloride, reacting at room temperature for 4 hours, adding 2M ammonium acetate solution for quenching reaction, preparing and purifying in a reversed phase manner, and freeze-drying to obtain a target compound B;
(1-3) suspending 2g of the compound B and 2eq of triphenylphosphine, 2eq of dithiodipyridine, 8eq of imidazole and 1eq of TEA in 20mL of DMF, reacting at room temperature for 8 hours, monitoring the reaction by HPLC until the raw materials are less than or equal to 1%, adding 4M sodium perchlorate acetone solution into the reaction solution, performing suction filtration, and fully washing the filter cake by acetone to obtain a target compound C;
(1-4) weighing 2g of the target compound C, suspending in 20mL of DMF, adding 3.5eq of triethylamine phosphate and 8eq of manganese chloride, stirring at room temperature for 8 hours, monitoring the reaction by HPLC (high performance liquid chromatography) until the raw material is less than or equal to 1%, and pouring the reaction liquid into 200mL of water to obtain a crude aqueous solution of the compound D. Slowly dropwise adding 8eq of dimethyl sulfate, adjusting the pH value to be not more than 5 by using 2M sodium hydroxide in the process, monitoring the reaction by using HPLC, and after the reaction is finished, purifying by using ion chromatography and freeze-drying to obtain a target compound E;
(1-5) suspending 1g of the compound E and 2eq of triphenylphosphine, 2eq of dithiodipyridine, 8eq of imidazole and 1eq of TEA in 10mL of DMF, reacting at room temperature for 8 hours, monitoring the reaction by HPLC until the raw materials are less than or equal to 1%, adding 4M sodium perchlorate acetone solution into the reaction solution, performing suction filtration, and fully washing the filter cake by acetone to obtain a target compound F;
the specific reaction scheme for intermediate F is shown in equation (1):
Figure BDA0003707070850000081
the synthetic route of A-G-P used in Synthesis example 1 was: weighing 5kg of 2 ' OMe-rA phosphoramidite monomer in a single-mouth bottle, dissolving with 50L of dichloromethane, adding 2.73kg of 2 ', 3 ' acetyl guanosine, cooling to 25 +/-2 ℃, adding 880g of tetrazole under the action of nitrogen blowing, and heating to 25 +/-2 ℃ for reaction. Monitoring the reaction, adding 1.2eq of iodopyridine solution into the reaction solution, performing spin drying after the monitoring reaction is finished, dissolving the concentrated ointment into 4L of dichloromethane, adding 1.1eq of trifluoroacetic acid, performing spin drying after the monitoring reaction is finished, pulping petroleum ether/dichloromethane according to a certain ratio, and filtering to obtain an intermediate G1; dissolving G1 in 4L acetonitrile, adding 1.2eq phosphine reagent and 1.2eq tetrazole, fully stirring for reaction, monitoring after the reaction is finished, adding 1.2eq iodopyridine solution into the reaction solution, monitoring after the reaction is finished, spin-drying, adding 3L methanol and 3L strong ammonia water into a spinner bottle, reacting for 4 hours at room temperature, monitoring for reaction, spin-drying after the reaction is finished, adding 20L ultrapure water, entering a reverse ion permeation device, washing, concentrating, and freeze-drying to obtain a target compound A-G-P, wherein the reaction route flow is as follows equation (2):
Figure BDA0003707070850000082
Figure BDA0003707070850000091
synthesis of morpholine-substituted A-G-P used in Synthesis example 2 referring to the A-G-P synthesis in example 1, the scheme of the morpholine-substituted A-G-P reaction scheme is as follows, equation (3):
Figure BDA0003707070850000092
wherein the preparation of C1 comprises the following steps: 1) 10g A1 and DMAP (0.1eq.) were suspended in 10 volumes of pyridine, and TBDPSCl (1.1eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was completed at room temperature. Concentrating the reaction liquid to remove pyridine, and recrystallizing the crude product with methanol to obtain A1; 2) wherein the synthesis of B1 refers to the synthesis step of intermediate a; 3) 5g B1 was dissolved in a mixed solvent of dichloromethane and acetonitrile (1: 1, 40mL), a solution of lithium bromide (3.2eq.) in acetonitrile (10mL) was added dropwise to the reaction solution at 0 ℃, and after stirring for 2 minutes, DBU (3.2eq.) was added dropwise and stirring was continued for 2 minutes. N, N-dimethylphosphoaminyldichloride (1.6eq.) was added dropwise to the reaction mixture, followed by reaction at room temperature for 1 hour and TLC to monitor completion of the reaction. The reaction solution was concentrated and purified by column chromatography to give C1.
Example 1: synthesis method of initial capped oligonucleotide primer containing morpholine ring structure by taking intermediate F and A-G-P as raw materials
Intermediate F (2mol) is dissolved in a solution containing MnCl 2 (20mol) in DMF and added to a solution of A-G-P (1.8mol) in DMF. The reaction was stirred at room temperature. After 24 hours, the reaction was stopped with 10L of 0.25M EDTA solution. The mixture was loaded onto a DEAE Sephadex column (30X 500 cm). The product was eluted using a linear gradient of 0-1.0M TEAB eluent. Collecting eluate with HPLC purity of more than 97%, concentrating the above separated solution, loading to strong anion resin, eluting with 0-1.0M sodium acetate eluate in linear gradient, collecting eluate with HPLC purity of more than 98.5%, mixing high purity eluates, and subjecting to nanofiltrationRemoving residual sodium acetate solution and concentrating to obtain the target product, wherein the reaction scheme is as follows, and the formula (4) is as follows:
Figure BDA0003707070850000101
example 2: synthesis method of initial capped oligonucleotide primer containing morpholine ring structure by using intermediate F and morpholine substituted A-G-P as raw materials
The starting capped oligonucleotide primers of example 2 were obtained by the synthesis method of the objective product of reference example 1 using intermediate F and morpholine-substituted A-G-P as starting materials. Scheme, equation (5) below:
Figure BDA0003707070850000102
example 3: synthesis method of initial capped oligonucleotide primer containing morpholine ring structure by using intermediate N and morpholine substituted A-G-P as raw materials
The starting capped oligonucleotide primers of example 2 were obtained by the synthesis method of the objective product of reference example 1 using intermediate N and morpholine-substituted A-G-P as starting materials. Scheme, equation (6) below:
Figure BDA0003707070850000103
wherein the intermediate N is obtained by the following steps: 1) weighing 5g of guanosine, dispersing the guanosine in 50mL of DMF, carrying out ice bath to ensure that the internal temperature of the reaction solution is lower than 10 ℃, adding 1.2eq of TBSCl in two batches, monitoring the reaction by HPLC (high performance liquid chromatography) until the raw material is less than or equal to 5%, adding 100mL of water after the reaction is finished to precipitate a product, filtering and washing a filter cake; dissolving 2g of filter cake in 10ml of trimethyl phosphate, cooling the reaction liquid to 0 ℃, slowly adding 1.2eq of phosphorus oxychloride dropwise, reacting at a low temperature for 4 hours, adding 2M of ammonium acetate solution, quenching, purifying by reverse phase chromatography to obtain a target compound f, fully reacting the obtained compound f with 1eq of triphenylphosphine, 2eq of dipyridyl disulfide and 4eq of imidazole, adding the reaction liquid into 4M of sodium perchlorate acetone solution, precipitating, and fully washing the filter cake with acetone to obtain a target compound g;
2) weighing 2g of target compound g, dissolving the target compound g in DMF, adding 3eq of tributylamine phosphate, fully stirring to obtain a target compound h, adding 20eq of aqueous solution into reaction liquid, cooling the reaction liquid to 4 ℃, slowly dropwise adding dimethyl sulfate, adjusting Ph to be not more than 5 by using 2M sodium hydroxide in the process, monitoring the reaction by HPLC, and purifying by ion chromatography after the reaction is finished to obtain a target compound i;
3) dissolving 4g of a compound i in 50mL of DMF, fully reacting with 1eq of triphenylphosphine, 2eq of dithiodipyridine and 4eq of imidazole, adding a reaction solution into 4M of a sodium perchlorate acetone solution, precipitating, and fully washing a filter cake with acetone to obtain a target compound N; scheme, equation (7) below:
Figure BDA0003707070850000111
comparative example 1: m7 GpppA 2’Ome pG
m7 GpppA 2’Ome pG Synthesis referring to the synthesis of the above examples, the scheme, equation (8) below:
Figure BDA0003707070850000112
the initial capped oligonucleotide primers containing morpholine ring structures obtained in each example and the capping analog structures obtained in comparative examples are shown in Table 2 below,
TABLE 2
Figure BDA0003707070850000121
Test example 1: determination of mRNA in vitro transcription yield and capping efficiency
In vitro synthesis of mRNA using an initial capped oligonucleotide primer containing a morpholine ring structure: firstly, carrying out NotI linearization on plasmids, and carrying out enzyme digestion at 4 ℃ overnight; extracting a DNA template; mRNA was synthesized by in vitro transcription using the initial capped oligonucleotide primers containing the morpholine ring structures of examples 1-3 and comparative example 1, respectively, as the cap structure.
The reaction system is shown in Table 3:
TABLE 3
System of Dosage of
T7 RNA polymerase 50U
10X buffer 2μl
100mM ATP 1μl
100mM GTP 1μl
100mM CTP 1μl
100mM N1-Me-pUTP 1μl
100mM cap analogs 1μl
Inorganic pyrophosphatase 0.05U
Nuclease inhibitors 20U
Sterile enzyme-free water Make up to 20. mu.l
Form panel 1μg
Remarking: in the experimental process, the volume of the materials needed by the system is calculated firstly, and then the sample is added. Firstly, adding sterile enzyme-free water into a system, then sequentially adding 10X buffer, NTPs and a cap analogue, mixing uniformly, then gently centrifuging, then adding a nuclease inhibitor, inorganic pyrophosphatase, T7 RNA polymerase and a linearized DNA template, fully mixing uniformly, gently centrifuging, and incubating at 37 ℃. After 2 hours DNase I1U was added and incubation continued at 37 ℃ for 30 minutes to remove DNA template and then RNA purification was performed, typically using magnetic bead purification. Purified mRNA was solubilized with sterile, enzyme-free water, followed by quantitative detection using Nanodrop One.
Liquid chromatography mass spectrometry (LC-MS) was used to detect IVT capping rates of mrnas of different starting cap analogs; firstly, a section of labeled DNA probe matched with mRNA starting base of a transcription product is required to be designed, the common label is labeled with biotin, streptavidin-labeled magnetic beads are washed and incubated with the synthesized DNA probe, mRNA and 10 XRNase H reaction buffer for 30 minutes at room temperature, the incubation and the slow mixing are carried out simultaneously, then 20ul RNase H (5U/ul) is added for incubation for 37 ℃ for 3 hours, and the mixing is carried out once every half hour. And (3) cleaning the magnetic beads after the incubation is finished, adding 100 mu L of 75% methanol heated to 80 ℃ into the cleaned magnetic beads, heating the mixture on a heating plate to 80 ℃, keeping the mixture for 3 minutes, then placing the mixture on a magnetic frame to absorb supernatant, and drying the supernatant at room temperature for 45 minutes to 10 mu L by using an evaporation centrifuge. The sample was then resuspended in 50. mu.l of 100. mu.M EDTA/1% MeOH and used for LC-MS analysis to determine the capping of the RNA in the transcription reaction. Since the capped and uncapped bases are clearly distinguished in molecular weight, the capping rate of mRNA transcription initiated by different cap analogs can be determined by using the difference in molecular mass. The specific results are shown in Table 4.
TABLE 4
Figure BDA0003707070850000131
Figure BDA0003707070850000141
As can be seen from the experimental results, the initial capping oligonucleotide primer containing a morpholino ring structure of the present application has the same level of mRNA in vitro transcription yield and capping efficiency as compared to the comparative example.
Test example 2: determination of the ability of mRNA to bind to RIG-I
RIG-I consists essentially of two repeated Caspase Activation and Recruitment Domains (CARDs) at the N-terminus, a helicase structure in the middle and a C-terminal RNA domain. The N-terminal CARD domain of RIG-I, even in the absence of viral infection, overexpresses the domain to promote secretion of type I Interferon (IFN) by cells, and therefore, this domain is primarily responsible for downstream signaling.
In this study, 293T cells were transfected with mRNA for eGFP, which was transcribed in vitro using the initial capped oligonucleotide primers containing the morpholino ring structures of examples 1-3 and the cap analogs of comparative examples 1-2 as initiators, and the cells were harvested after 24 hours, and the intracellular protein RIG-I was co-immunoprecipitated with its associated RNA using the RNA co-immunoprecipitation method, and finally these mRNAs were subjected to reverse transcription and real-time quantitative PCR.
The specific culture conditions of the cells are the same as those above, the cells are collected after 24h of transfection, firstly, a fixing solution is added for incubation, glycine solution with proper concentration is added after 10min to stop the reaction, and the cells are collected. The collected cells were lysed with the lysate, centrifuged at 12000rpm at 4 ℃ for 10min, and the supernatant was incubated with RIG-I or IgG antibody, respectively, overnight with a shaker at 4 ℃. And then adding 20 mu l of Protein A/G magnetic beads, incubating for 4h at 4 ℃, washing on a magnetic frame, and extracting RNA after washing is finished, wherein the RNA can be used for verifying the expression result by subsequent RT-qPCR. The binding ability of the different cap analogue nucleotide mRNAs to RIG-1 results are given in Table 5 below:
TABLE 5
Different cap analogs Ability to bind to RIG-I
Example 1 1.5±0.11
Example 2 2.1±0.32
Example 3 2.5±0.33
Comparative example 1 3.2±0.52
As can be seen from the experimental data in Table 4 above, the initial capped oligonucleotide primer containing morpholine ring structure in the present invention has significantly lower immunogenicity when applied to mRNA synthesizing cells than the comparative example Cleancap.
Test example 3: intracellular translation Effect detection of mRNA
The different mRNA products obtained from IVT were transfected into 293T cells. 293T cells were plated (24-well plates) at (0.5-1). times.105 cells, and transfection experiments with cells within 50 passages were recommended. Require that the cells be subcultured 24 hours before transfectionThe addition of antibiotics has no effect on the transfection effect. The cell density is generally 60-80% and 2. mu.g of mRNA per well is transfected, and the Transfection Reagent is Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) and is used according to the method of use. The transfected cells were placed at 37 ℃ CO 2 In the incubator, the medium is replaced with fresh complete medium 4-6 hours after transfection. CO at 37 deg.C 2 After incubation in the incubator for 24 hours, fluorescence microscope was used to observe the fluorescence intensity of GFP therein. The results are shown in FIGS. 1 and 2, and it is apparent from the results that the expression efficiency of the mRNA of the present invention is significantly higher than that of the comparative example, while neither of them causes significant cell death.
This result indicates that the initial capped oligonucleotide primer containing the morpholine ring structure of example 1 of the present application has higher expression efficiency; that is, the effective protein translation efficiency of the initial capped oligonucleotide primer containing the morpholine ring nucleoside structure applied to mRNA synthesis is obviously higher than that of the cap structure of Cleancap (comparative example 1).
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (3)

1. An initial capping oligonucleotide primer comprising a morpholino ring structure, comprising the structure:
Figure FDA0003707070840000011
wherein, X 1 、X 2 And X 3 Are each independently O, CH 2 Or NH;
Y 1 、Y 2 and Y 3 Each independently is O, S, Se or BH 3 ；
R a Is composed of
Figure FDA0003707070840000012
Rb is
Figure FDA0003707070840000013
And when Ra is
Figure FDA0003707070840000014
When Rb is
Figure FDA0003707070840000015
R 1 Is hydrogen, hydroxy, substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted N-dihydrocarbyl, 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;
R 2 and R 3 Independently H, OH, alkyl, O-alkyl, halogen;
B 1 and B 2 Independently, a natural, or modified, or non-natural nucleobase.
2. The method of claim 1, comprising the steps of: (1) synthesis of intermediate F: synthesizing a compound A from guanosine, and successively carrying out diphosphorylation, methylation of N7 and imidization of polyphosphoric acid on the basis of the compound A to synthesize an intermediate F; (2) preparation of a phosphate-linked dinucleotide: coupling a phosphoramidite monomer and a disubstituted nucleoside monomer under the action of tetrazole to form a first phosphate ester bond, removing a protecting group through acid action, introducing a second phosphoric acid, and finally hydrolyzing to obtain a dinucleotide connected with the phosphate ester bond; (3) synthesis of initial capped oligonucleotide primers containing a morpholine ring structure: reacting the intermediate F with dinucleotide connected with a phosphate bond to prepare an initial capping oligonucleotide primer containing a morpholine ring structure;
the phosphoramidite monomer has a structural formula as follows:
Figure FDA0003707070840000021
wherein R is 4 H, OH, alkyl, O-alkyl, halogen; b is 3 And B 4 Independently, a natural, or modified, or non-natural nucleobase.
3. The use of the initial capped oligonucleotide primer comprising a morpholino ring structure of claim 1 wherein: the mRNA of the initial capped oligonucleotide primer containing the morpholine ring structure was capped using an IVT reaction system using T7 RNA polymerase.
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