CN114853836B - Initial capping oligonucleotide primer containing GNA structure and preparation method and application thereof - Google Patents

Initial capping oligonucleotide primer containing GNA structure and preparation method and application thereof Download PDF

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CN114853836B
CN114853836B CN202210732340.8A CN202210732340A CN114853836B CN 114853836 B CN114853836 B CN 114853836B CN 202210732340 A CN202210732340 A CN 202210732340A CN 114853836 B CN114853836 B CN 114853836B
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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 capping oligonucleotide primer containing a GNA structure, a preparation method and application thereof, wherein the molecular formula of the initial capping oligonucleotide primer containing the GNA structure is m7 GpppApG, m7 G is N 7 -methylated guanosine or any guanosine analogue, and A is any natural, modified or unnatural adenosine or any adenosine analogue. The mRNA synthesized by the initial capping oligonucleotide primer containing the GNA structure can obviously reduce the expression level of cell inflammatory factors and has lower immunogenicity.

Description

Initial capping oligonucleotide primer containing GNA structure and preparation method and application thereof
Technical Field
The invention relates to the technical field of chemistry and bioengineering, in particular to an initial capping oligonucleotide primer containing a GNA 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, and capping of the mRNA promotes its normal function in the cell. Synthesis of mRNA by 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, enabling workers to prepare RNA molecules that perform properly in a variety of biological applications. Such applications include research applications and commercial production of polypeptides, for example, in cell-free translation systems to produce polypeptides comprising "unnatural" amino acids at specific sites, or in cultured cells to produce polypeptides that require post-translational modification for their activity or stability. In the latter system, synthesis proceeds for a significantly longer time and thus more protein is produced.
Patent CN201680067458.6 reports compositions and methods for synthesizing 5' -capped RNAs. Wherein the starting capped oligonucleotide primer has the general form m 7Gppp[N2'OMe]n[N]m, wherein m 7 G is N 7 -methylated guanosine or any guanosine analogue, N is any natural, modified or non-natural nucleoside, "N" may be any integer from 0 to 4 and "m" may be an integer from 1 to 9. Cleancap belongs to Cap1, and CleanCap uses a trimer (m 7 GpppAmG) to initiate T7 transcription, as opposed to ARCA which uses a dimer (m 7 GpppG) to initiate T7 transcription. The method has higher yield, 4mg of capped RNA is prepared per milliliter of transcription reaction system, the capping efficiency can reach 90 percent, and the immunogenicity of the transcription product is lower than that of ARCA. At present, the problem of immunogenicity of mRNA remains an important point in this research field. The 5' cap structure is a key structure for reducing immunogenicity, so that more cap structures are required to be designed for better immunogenicity reduction.
Capping analogs at the 5' position are key structures for reducing mRNA immunogenicity, and by optimizing the structure of the cap analogs, the level of expression of cytokines can be reduced. Accordingly, there is a continuing need to optimize the structure of cap analogs and to find novel cap analogs that are less immunogenic and have lower levels of expression of cytokines than currently available Cleancap.
Disclosure of Invention
The application provides an initial capping oligonucleotide primer containing a GNA structure, a preparation method and application thereof, wherein the initial capping oligonucleotide primer containing the GNA structure contains the GNA structure which replaces the original five-membered sugar ring structure, and after replacement, the induced inflammatory factor expression can be reduced due to weak protein binding capacity of the GNA and an inflammatory pathway. Meanwhile, mRNA synthesized by cap analogues of GNA structure has the advantages of obviously reducing the expression level of cell inflammatory factors and lower immunogenicity.
An initial capped oligonucleotide primer comprising a GNA structure comprising the structure:
wherein, X 1、X2 and X 3 are respectively and independently O, CH 2 or NH;
Y 1、Y2 and Y 3 are each independently O, S, se or BH 3;
Ra is R b is/>And when Ra is/>In the time-course of which the first and second contact surfaces,
Rb isWhen Ra is/>When Rb is/>
R 1、R2、R3 is independently H, OH, alkyl, O-alkyl, halogen;
R 4 is hydrogen, hydroxy, substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted N-dialkyl, 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、B2 and B 3 are independently natural, or modified, or unnatural nucleobases.
A method of preparing an initial capped oligonucleotide primer comprising a GNA structure, comprising the steps of: (1) Synthesis of imidazole diphosphate intermediate (Compound 21): n2-iso Ding Xiandiao purine (compound 14) is used as a starting material to sequentially carry out reactions such as glycosylation, diphosphate, methylation of N7, imidization of polyphosphoric acid and the like to synthesize an imidazole diphosphate intermediate; (2) preparation of phosphoester-linked dinucleotides: coupling a phosphoramidite monomer and a disubstituted nucleoside monomer under the action of tetrazole to form a first phosphoester bond, removing a protecting group under the action of acid, introducing a second phosphoric acid, and finally hydrolyzing to obtain a dinucleotide connected with the phosphoester bond; (3) Synthesis of initial capped oligonucleotide primers containing GNA structures: reacting the imidazole diphosphate intermediate with a phosphate-linked dinucleotide to prepare an initial capped oligonucleotide primer containing a GNA structure;
The structural formula of the phosphoramidite monomer is as follows:
Wherein R 5 is H, OH, alkyl, O-alkyl, halogen; b 4 is a natural, or modified, or unnatural nucleobase.
The above-mentioned disubstituted nucleoside monomer is selected fromAny one of them.
The preparation method of the initial capping oligonucleotide primer containing the GNA structure specifically comprises the following steps:
(1) Synthesis of Compound 21:
(1-1) weighing N2-iso Ding Xiandiao purine (compound 14), dissolving in acetonitrile, stirring uniformly, clarifying, adding diacetyl protected glycerin analogue, fully mixing, adding BSA and stannic chloride, reacting at room temperature for 5 hours, monitoring the reaction by HPLC, adding saturated ammonium chloride solution after the reaction is finished to quench the reaction, extracting the reaction system with ethyl acetate for three times, drying the ethyl acetate phase, and separating by a column to obtain a product 15;
(1-2) weighing a compound 15, dissolving in methanol, adding ammonia water, spinning to dry a system after the reaction is finished, adding acetonitrile and pyridine after the water content is less than 100ppm and the acetonitrile and pyridine are added, slowly dropwise adding trimethylchlorosilane until the temperature of the reaction system is less than 4 ℃, adding benzoyl chloride after the dropwise addition is finished, reacting for 5 hours at room temperature, and concentrating a target compound 16;
(1-3) Compound 16 was dissolved in pyridine, tsCl was added to the reaction mixture in portions under ice bath, and then the mixture was allowed to warm to room temperature for reaction. TLC monitored the reaction to completion. Concentrating the reaction solution, and purifying the crude product by column chromatography to obtain a compound 17;
(1-4) Compound 17 was dissolved in DMF, and ammonium tris (tetrabutyl) pyrophosphate was added to the reaction solution, followed by reaction at room temperature for 20 hours. HPLC monitored reaction was complete. Concentrating under reduced pressure to remove most of the solvent to obtain crude product of the compound 18. The crude compound 18 was dissolved in concentrated aqueous ammonia and stirred at room temperature for 2 hours. HPLC monitored reaction was complete. Concentrating to remove ammonia water, and diluting with deionized water. Subjecting the mixture to DEAE Sephadex, and linear gradient elution of mobile phase with TEAB eluent of 0-1.0M to obtain compound 19;
(1-5) weighing the compound 19, dissolving in an aqueous solution, cooling the reaction solution to 4 ℃, slowly dropwise adding dimethyl sulfate, adjusting the 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 compound 20;
(1-6) Compound 20 was dissolved in DMF and reacted with triphenylphosphine, dithiodipyridine, imidazole, the reaction mixture was precipitated by adding 4M sodium perchlorate in acetone, and the cake was washed thoroughly with acetone to give the objective compound 21.
(2) Preparation of phosphoester-linked dinucleotides:
And (3) weighing phosphoramidite monomer, dissolving in methylene dichloride, adding disubstituted nucleoside monomer, 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, spin-drying after the monitoring reaction is finished, dissolving the concentrated ointment into dichloromethane, adding trifluoroacetic acid, spin-drying after the monitoring reaction is finished, pulping petroleum ether/dichloromethane according to a certain proportion, and filtering to obtain an intermediate; dissolving the intermediate in trimethyl phosphate, adding phosphorus oxychloride, stirring thoroughly for reaction, adding methanol and concentrated ammonia water into a rotary bottle after the reaction is monitored, reacting for 4 hours at room temperature, monitoring the reaction, spin-drying after the reaction is finished, adding ultrapure water, entering reverse ion permeation equipment, washing, concentrating, and freeze-drying to obtain a target compound;
(3) Synthesis of initial capped oligonucleotide primers containing GNA structures:
Compound 21 was dissolved in DMF solution containing MnCl 2 (0.2 mol) and added to DMF solution of the phosphoester-linked dinucleotide. The reaction was stirred at room temperature. After 24 hours, the reaction was stopped with 0.25M EDTA solution. The mixture was loaded onto DEAESephadex columns (30X 500 cm). The product was eluted using a linear gradient of 0-1.0M TEAB eluent. Collecting the eluted product with HPLC purity of > 97%, concentrating the above separated liquid, loading to strong anion resin, linearly gradient eluting with 0-1.0M sodium acetate eluent, collecting the eluted product with HPLC purity of > 98.5%, mixing the high purity eluates, removing residual sodium acetate solution by nanofiltration equipment, and concentrating to obtain the target product containing initial capping oligonucleotide primer with GNA structure.
The initial capping oligonucleotide primer containing the GNA structure is used for mRNA capping under a T7 RNA polymerase system. T7 RNA polymerase is a DNA dependent RNA polymerase with high specificity for phage T7 promoter sequences. The enzyme synthesizes a large amount of RNA from DNA inserted downstream of the T7 promoter into the transcription vector. The T7 RNA polymerase catalyzed IVT (in vitro transcription) reaction system is the most mature mRNA preparation system at present.
Typically, 20ul of IVT reaction system contains 50U of T7 RNA polymerase, and the best transcription yield and capping efficiency can be obtained with 1ul of cap analogue (100 mM).
The invention provides an initial capping oligonucleotide primer containing a GNA structure, a preparation method and application thereof, wherein the initial capping oligonucleotide primer containing the GNA structure is suitable for mRNA (messenger ribonucleic acid) produced by taking a DNA sequence as a template and utilizing an in-vitro co-transcription method, the DNA sequence can be derived or modified from virus, animal, plant and other species, and meanwhile, 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 analogues Cleancap, the initial capping oligonucleotide primer containing the GNA structure has the advantages that mRNA produced by the initial capping oligonucleotide primer has obviously reduced expression level of cell inflammatory factors and lower immunogenicity.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The raw material names and sources used in each example are shown in table 1 below:
TABLE 1
Example 1: synthesis method of initial capping oligonucleotide primer containing GNA structure by taking compound 9 and compound N as raw materials
Compound N was dissolved in DMF solution containing MnCl 2 (0.2 mol) and added to DMF solution of compound 9 (1.8 mol). 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 DEAESephadex columns (30X 500 cm). The product was eluted using a linear gradient of 0-1.0M TEAB eluent. Collecting the eluted product with HPLC purity of more than 97%, concentrating the separated liquid, loading the concentrated product into strong anion resin, linearly gradient eluting with 0-1.0M sodium acetate eluent, collecting the eluted product with HPLC purity of more than 98.5%, combining the high-purity eluent, removing residual sodium acetate solution by nanofiltration equipment and concentrating to obtain target product GNA-1, and the reaction route flow is as shown in the following equation (4):
Wherein, the compound 9 is prepared by the following steps:
(1-1) weighing 50g of compound 1, dissolving in 200ml of acetonitrile, stirring uniformly, clarifying, adding 1.2eq of diacetyl-protected glycerol analogue, fully mixing, adding 1.2eq of BSA and 1.4eq of tin tetrachloride, reacting for 5 hours at room temperature, monitoring the reaction by HPLC, adding 300ml of saturated ammonium chloride solution after the reaction is finished, quenching the reaction, extracting the reaction system for three times by using ethyl acetate, drying the ethyl acetate phase, and separating by a column to obtain a product 2;
(1-2) weighing 10g of compound 2, dissolving in 20ml of methanol, adding 2eq of ammonia water, carrying out a spin-drying system after the reaction is finished, adding 2 volumes of acetonitrile with water, adding 2 volumes of acetonitrile after the water content is less than 100ppm, 2eq of pyridine, slowly dropwise adding 2eq of trimethylchlorosilane in ice bath until the temperature of the reaction system is less than 4 ℃, adding 1.2eq of benzoyl chloride after the dropwise addition is finished, reacting for 5 hours at room temperature, concentrating the target compound 3, and obtaining the target compound 4 after purification by reacting with tetrazole and phosphine reagents;
(1-3) 50g of 4 phosphoramidite monomer was weighed into a single-mouth bottle, dissolved with 200mL of methylene chloride, then 1.2eq of 2',3' acetyl guanosine was added, the temperature was lowered to 25+ -2deg.C, 0.5eq of tetrazole was added under nitrogen bubbling, and the temperature was raised to 25+ -2deg.C for reaction. After the monitoring reaction is finished, adding 1.2eq of iodopyridine solution into the reaction solution, spin-drying after the monitoring reaction is finished, dissolving the concentrated ointment into 400mL of dichloromethane, adding 1.1eq of trifluoroacetic acid, spin-drying after the monitoring reaction is finished, pulping petroleum ether/dichloromethane according to a certain proportion, and filtering to obtain a compound 7; dissolving a compound 7 in 400mL of trimethyl phosphate, adding 1.2eq of phosphorus oxychloride, fully stirring for reaction, adding 300mL of methanol and 300mL of concentrated ammonia water into a rotary bottle after monitoring the reaction, reacting for 4 hours at room temperature, monitoring the reaction, spin-drying after the reaction, adding 20L of ultrapure water, entering reverse ion permeation equipment, washing, concentrating, and freeze-drying to obtain a target compound 9;
The specific scheme for compound 9 is as follows equation (1):
Wherein, the compound N is obtained by the following steps: 1) Weighing 5g of guanosine, dispersing in 50mL of DMF, carrying out ice bath to enable the internal temperature of the reaction liquid to be lower than 10 ℃, adding 1.2eq of TBSCl into the reaction liquid in two batches, monitoring the reaction until the raw material is less than or equal to 5% by HPLC, 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 solution to 0 ℃, slowly dropwise adding 1.2eq phosphorus oxychloride, reacting for 4 hours at low temperature, adding 2M ammonium acetate solution to quench the reaction, purifying by reverse phase chromatography to obtain a target compound f, fully reacting the obtained compound f with 1eq triphenylphosphine, 2eq dithiodipyridine and 4eq imidazole, adding the reaction solution into 4M sodium perchlorate acetone solution to precipitate, and fully washing the filter cake with acetone to obtain the target compound g;
2) Weighing 2g of target compound g, dissolving DMF, adding 3eq of tributylamine phosphate, fully stirring to obtain target compound h, adding 20eq of aqueous solution into the reaction solution, cooling the reaction solution to 4 ℃, slowly dropwise adding dimethyl sulfate, regulating Ph with 2M sodium hydroxide to be not more than 5 in the process, monitoring the reaction by HPLC, and purifying by ion chromatography after the reaction is finished to obtain target compound i;
3) 4g of compound i is dissolved in 50mL of DMF and fully reacted with 1eq of triphenylphosphine, 2eq of dithiodipyridine and 4eq of imidazole, the reaction solution is added into 4M sodium perchlorate acetone solution to be separated out, and the filter cake is fully washed by acetone to obtain the target compound N.
Compound N scheme, equation (7) below:
Example 2: synthesis method of initial capping oligonucleotide primer containing GNA structure by using compound 21 and A-G-P as raw materials
The starting capped oligonucleotide primer GNA-2 of example 2 was obtained by the method of synthesizing the target product GNA-1 of reference example 1, starting from compound 21 and A-G-P. Scheme of reaction scheme, equation (5) below:
Wherein, the preparation of the compound 21 comprises the following steps: 1) Synthesis of compound 15 reference compound 2; 2) Synthesis of compound 16 reference compound 3; 3) 10g of compound 16 was dissolved in pyridine (100 mL), tsCl (1.2 eq.) was added to the reaction solution in portions under ice bath, and then the mixture was warmed to room temperature for reaction. TLC monitored the reaction to completion. Concentrating the reaction solution, and purifying the crude product by column chromatography to obtain a compound 17; 4) 5g of compound 17 was dissolved in DMF (50 mL), and ammonium tris (tetrabutyl) pyrophosphate (2.0 eq.) was added to the reaction solution, followed by reaction at room temperature for 20 hours. HPLC monitored reaction was complete. Concentrating under reduced pressure to remove most of the solvent to obtain crude product of the compound 18. The crude compound 18 was dissolved in 150mL of concentrated aqueous ammonia and stirring was continued at room temperature for 2 hours. HPLC monitored reaction was complete. Concentrating to remove ammonia water, and diluting with deionized water. Subjecting the mixture to DEAE Sephadex, and linear gradient elution of mobile phase with TEAB eluent of 0-1.0M to obtain compound 19; 5) Synthesis of compound 20 reference compound 11; 6) Synthesis of Compound 21 reference the step of synthesizing Compound 12.
Scheme of compound 21, equation (3) below:
Wherein, the synthetic route of A-G-P is as follows: 5kg of 2' OMe-rA phosphoramidite monomer is weighed into a single-mouth bottle, dissolved by 50L of dichloromethane, added with 2.73kg of 2',3' acetyl guanosine, cooled to 25+/-2 ℃, added with 880g of tetrazole under nitrogen blowing, and heated to 25+/-2 ℃ for reaction. After the monitoring reaction is finished, adding 1.2eq of iodopyridine solution into the reaction solution, spin-drying after the monitoring reaction is finished, dissolving the concentrated ointment into 4L of dichloromethane, adding 1.1eq of trifluoroacetic acid, spin-drying after the monitoring reaction is finished, pulping petroleum ether/dichloromethane according to a certain proportion, and filtering to obtain a compound G1; dissolving G1 in 4L acetonitrile, adding 1.2eq of phosphine reagent and 1.2eq of tetrazole, fully stirring for reaction, adding 1.2eq of iodopyridine solution into the reaction liquid after monitoring the reaction, spin-drying after monitoring the reaction, adding 3L of methanol and 3L of concentrated ammonia water into a spin bottle, reacting for 4 hours at room temperature, monitoring the reaction, spin-drying after the reaction, adding 20L of ultrapure water, entering reverse ion permeation equipment, washing, concentrating, and freeze-drying to obtain a target compound A-G-P, wherein the reaction route flow is as shown in the following equation (2):
Example 3: synthesis method of initial capping oligonucleotide primer containing GNA structure by using compound 9 and compound 21 as raw materials
Starting from compound 9 and compound 21 (compound 9 obtained by the preparation method in reference to example 1 and compound 21 obtained by the preparation method in reference to example 2), the synthesis method of the target product GNA-1 in reference to example 1 gives the starting capped oligonucleotide primer GNA-3 of example 3. Scheme of reaction scheme, equation (5) below:
comparative example 1: m7GpppA2'OmepG
m7GpppA2'Ome The synthesis of pG is referred to the synthesis of the above examples (the starting materials used refer to the preparation in each example), the scheme of the reaction scheme is as follows equation (8):
The initial capped oligonucleotide primers containing the GNA structure obtained in each example and the capped analogs obtained in the comparative examples are shown in Table 2 below,
TABLE 2
Test example 1: determination of mRNA in vitro transcription yield and capping efficiency
In vitro synthesis of mRNA using initial capped oligonucleotide primers containing GNA structures: linearizing the plasmid with NotI, and enzyme cutting at 4 ℃ overnight; extracting a DNA template; mRNA was synthesized by in vitro transcription using the initial capping oligonucleotide primers containing the GNA structure of examples 1-3 and comparative example 1, respectively, as the cap structure.
The reaction system is shown in Table 3:
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 analogue 1μl
Inorganic pyrophosphatase 0.05U
Nuclease inhibitors 20U
Sterile water Make up to 20. Mu.l
Template 1μg
Remarks: in the experimental process, the volume of materials required by the system is calculated first, and then the sample is added. Firstly, adding sterile and sterile water into a system, then sequentially adding 10 Xbuffer, NTPs and cap analogues, mixing uniformly, lightly centrifuging, then adding nuclease inhibitor, inorganic pyrophosphatase, T7 RNA polymerase and linearized DNA template, fully mixing uniformly, lightly centrifuging, and incubating at 37 ℃. DNase I1U was added after 2 hours and incubation was continued for 30 minutes at 37℃to remove the DNA template, followed by RNA purification, typically using a magnetic bead purification method. Purified mRNA was solubilized with sterile, aqueous water and subsequently quantitatively detected using a Nanodrop One.
Liquid chromatography mass spectrometry (LC-MS) was used to detect IVT capping rates of mRNA of different starting cap analogs; firstly, a section of DNA probe with a label matched with the initial base of mRNA of a transcription product needs to be designed, a general label is a biotin label, a streptavidin-labeled magnetic bead is washed and then incubated with the synthesized DNA probe, mRNA and 10X RNase H reaction buffer for 30 minutes at room temperature, the DNA probe, mRNA and 10X RNase H reaction buffer are slowly mixed while being incubated, and then 20ul RNase H (5U/ul) is added for incubation for 3 hours at 37 degrees, and the mixture is mixed every half hour. After the incubation, the beads were washed, 100. Mu.L of 75% methanol heated to 80℃was added to the washed beads, the mixture was heated to 80℃on a hot plate, kept for 3 minutes, and the supernatant was then sucked up on a magnetic rack and dried at room temperature for 45 minutes to 10. Mu.L 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 RNA capping during transcription. Since the capped and uncapped bases are significantly different in molecular weight, the difference in molecular mass can be used to determine the capping rate of mRNA transcription initiated by different cap analogs. The specific results are shown in Table 4.
TABLE 4 Table 4
Numbering device Yield (μg) Capping Rate (%)
Example 1 55 93.2
Example 2 50 91.3
Example 3 48 85.4
Comparative example 1 56 92.0
From the experimental results, it is understood that the initial capping oligonucleotide primers containing GNA structure of example 1 and example 2 of the present application have the same level of mRNA in vitro transcription yield and capping efficiency as compared with the comparative example; however, the capping efficiency of example 3 is significantly reduced, which may be disadvantageous for the catalytic reaction of T7 RNA polymerase due to the large gap between the structure and the cap analogue, resulting in reduced capping efficiency.
Test example 2: mRNA stimulates expression of inflammatory factors in cells
HeLa cells were plated in 6-well plates at a density of 4X 10 5/well and transfected at a cell density of approximately 80%. 2ug mRNA was transfected per well with Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) as transfection reagent and the transfection procedure was performed as described herein. After 24 hours cells were harvested, RNA was extracted using TRIzol and reverse transcribed into cDNA. Finally, detecting the expression of inflammatory factors in cells by using real-time quantitative fluorescence PCR, wherein the internal reference gene is beta-ACTIN. The detection of each gene was repeated three times, and the expression result of each gene was a relative value with respect to the result of the cap analogue of comparative example 1. Data are expressed as mean ± standard deviation, the results obtained are given in table 5 below:
TABLE 5
Numbering device Expression of IL-6 Expression of INFB Expression of INFA Expression of MX1
Example 1 0.5 0.6 0.6 0.7
Example 2 0.8 0.5 0.6 0.7
Example 3 1.1 1.3 1.2 1.0
Comparative example 1 1.0 1.0 1.0 1.0
From the experimental data in table 4 above, it can be seen that the initial capping oligonucleotide primers of examples 1 and 2 containing GNA structures of the present application have significantly reduced expression levels of cytokines and have lower immunogenicity compared to comparative example 1. However, example 3 showed higher levels of cytokine expression than comparative example 1, probably due to the less efficient capping of the capping analog, and the remaining uncapped mRNA in the product, resulting in enhanced immunogenicity.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein 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 capped oligonucleotide primer comprising a GNA structure, comprising the structure:
，
wherein, X 1、X2 and X 3 are respectively independent O;
Y 1、Y2 and Y 3 are each independently O;
Ra is Or/>Rb is/>Or/>And when Ra is/>When Rb is; When Ra is/>When Rb is/>；
R 1、R2、R3 is H, OH or halogen independently;
r 4 is hydrogen, hydroxy, methoxy;
b 1、B2 and B 3 are independently natural nucleobases.
2. The GNA structure-containing starting capped oligonucleotide primer of claim 1, wherein said primer is selected from any one of the following structures:
，
。
3. Use of an initial capped oligonucleotide primer comprising a GNA structure according to claim 1, wherein: the mRNA of the initial capping oligonucleotide primer containing GNA structure was capped using T7 RNA polymerase.
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