Cap2-mrna synthesis reagent composition, kit and use thereof

Abstract

A Cap2-mRNA synthesis reagent composition, a kit and the use thereof. Specifically, provided is an in-vitro co-transcriptional RNA capping reagent composition, which comprises: a Cap2 cap analog, magnesium chloride and a DNA template. By using the Cap2 cap analog, no competitive inhibition is formed with GTP, which increases the utilization rate of the cap analog. Thus, the mRNA synthesized by means of a transcription reaction has high capping efficiency, high yield and high integrity.

Description

Cap2-mRNA synthesis reagent composition, kit and application thereof TECHNICAL FIELD

[0001] The present application relates to a Cap2-mRNA synthesis reagent composition, kit and application thereof. BACKGROUND

[0002] In vitro transcription synthesis mRNA is to use RNA polymerase to transcribe and synthesize mRNA in vitro in a cell-free system with plasmid DNA as a template, and to add 5' cap and 3' poly(A) tail and other modifications to simulate the process of in vivo mRNA synthesis. In the process of in vitro transcription synthesis mRNA, the mRNA capping process is a very critical step. The cap structure of mRNA can be completed by post-transcriptional enzyme capping modification using capping enzyme and 2-O-methyltransferase, or by co-transcriptional capping with cap analogs.

[0003] Using co-transcriptional capping, cap analogs and GTP compete with each other as the starting sequence of mRNA at the start of transcription, so the molar ratio of cap analogs to GTP is an important factor affecting the capping rate in co-transcriptional capping synthesis mRNA.

[0004] The commonly used cap analog is Cap1 structure, however, there are multiple active groups on the nucleotides of Cap1 structure cap analogs, which need to be protected and deprotected multiple times during synthesis, resulting in low mRNA synthesis efficiency. SUMMARY

[0005] An object of the present application is to provide an improved in vitro transcription reaction system containing a structural cap analog.

[0006] Another object of the present application is to provide a method for preparing mRNA using a Cap2 structure cap analog.

[0007] Another object of the present application is to provide a kit for preparing 5' capped mRNA using a Cap2 structure cap analog.

[0008] In one aspect, the present application provides an in vitro co-transcriptional RNA capping reagent composition, comprising: a Cap2 cap analog, magnesium chloride, and a DNA template.

[0009] The in vitro co-transcriptional RNA capping reagent composition of the present application contains a Cap2 structure cap analog, which is used for in vitro transcription synthesis of 5' capped mRNA, and the capping efficiency of mRNA synthesized by transcription reaction is high, the yield is high, and the integrity is high.

[0010] According to specific embodiments of the present application, in the in vitro co-transcriptional RNA capping reagent composition of the present application, the Cap2 cap analog has the following structure of Formula I:

[0011] In Formula I:

[0012] R1is selected from any one of -H, -OH, C 1-6 alkyl and C 1-6 alkoxy;

[0013] R2is selected from any one of -H, -OH, halogen, C 1-6 alkoxy;

[0014] R3, R0are each independently selected from any one of F, Cl, Br, I;

[0015] J1, J2are each independently selected from a natural or modified pyrimidine nucleotide base, a natural or modified purine nucleotide base.

[0016] According to specific embodiments of the present application, in the in vitro co-transcriptional RNA capping reagent composition of the present application, the Cap2 cap analog is selected from one or more of the following compounds:

[0017] According to specific embodiments of the present application, in the in vitro co-transcriptional RNA capping reagent composition of the present application, the Cap2 cap analog can be synthesized according to the method described in CN202310391954.9, the contents of which are hereby incorporated by reference in their entirety.

[0018] According to specific embodiments of the present application, in the in vitro co-transcriptional RNA capping reagent composition of the present application, it can further comprise one or more of nucleotide triphosphate molecules, RNA polymerase and other common in vitro co-transcriptional RNA capping reagents. Preferably, the in vitro co-transcriptional RNA capping reagent composition of the present application further comprises one or more of RNAse inhibitors, inorganic pyrophosphatase, buffers. Preferably, the in vitro co-transcriptional RNA capping reagent composition of the present application can further comprise one or more of DNAse, LiCl precipitation solution. The specific selection and amount of the reagents such as nucleotide triphosphate molecules, RNA polymerase, RNAse inhibitors, inorganic pyrophosphatase, buffers, DNAse, LiCl precipitation solution can be determined according to the common reagents and amounts used in the in vitro co-transcriptional RNA capping process in the prior art.

[0019] In another aspect, the present application also provides an in vitro co-transcriptional RNA capping kit comprising the aforementioned in vitro co-transcriptional RNA capping reagent composition of the present application.

[0020] In another aspect, the present application also provides the use of the aforementioned in vitro co-transcriptional RNA capping reagent composition or the aforementioned kit in the preparation of 5' capped RNA.

[0021] In another aspect, the present application also provides an in vitro co-transcriptional RNA capping method, which comprises performing template transcription in a transcription reaction system comprising the aforementioned in vitro co-transcriptional RNA capping reagent composition of the present application to synthesize 5' capped RNA.

[0022] According to a specific embodiment of the present application, the RNA is mRNA.

[0023] According to a specific embodiment of the present application, in the in vitro co-transcriptional RNA capping method of the present application, the concentration of magnesium chloride in the transcription reaction system is 15 mM to 40 mM, preferably 20 mM to 30 mM.

[0024] According to a specific embodiment of the present application, in the in vitro co-transcriptional RNA capping method of the present application, the concentration of Cap2 cap analog in the transcription reaction system is ≥ 1.5 mM, preferably the concentration is 1.5 mM to 10 mM, more preferably 2.5 mM to 10 mM.

[0025] According to a specific embodiment of the present application, in the in vitro co-transcriptional RNA capping method of the present application, the transcription reaction temperature is 36.0℃-43.5℃, preferably 40.0℃-43.5℃, more preferably 42.0℃-42.7℃.

[0026] The Cap2 cap analog used in the present application does not form competitive inhibition with GTP, thereby improving the utilization rate of the cap analog, and the mRNA synthesized by the transcription reaction has high capping efficiency, high yield and high integrity. There is a complete in vitro transcription reaction system. The input amount of cap analog is reduced, and the material cost in the production process of capped mRNA is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is an agarose gel electrophoresis detection diagram of the capped mRNA prepared in Example 1.

[0028] Figure 2 shows the effect of mRNA-LNP formed by mRNA with different capping methods on the specific binding of gE antibody to CD4+ cells and the expression level of cytokines. DETAILED DESCRIPTION

[0029] Before particular embodiments of the present application are described, it is to be understood that the application is not limited to the particular specific embodiments described herein; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting as the scope of the present application.

[0030] When a numerical range is given in the embodiments, it is understood that, unless otherwise stated, each numerical range's two endpoints and any number in between can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Except in the Examples, or where otherwise explicitly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, particular methods, devices, and materials are now described, in order to most clearly demonstrate the embodiments of the present application.

[0031] Unless otherwise stated, the experimental methods, detection methods, preparation methods disclosed in the present application all use the conventional techniques in the art.

[0032] For the purposes of the present application, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0033] In this specification, references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", "implementations", etc. mean that the particular element(s) described is included in at least one embodiment of the application, and can or can not be present in other embodiments. In addition, it is to be understood that the described elements can be combined in any suitable manner in the various embodiments.

[0034] In each of the embodiments, the general instrument reagent information is shown in Table 1 and Table 2.

[0035] Table 1, Instrument Information

[0036] Table 2, Material Source Information

[0037] In each of the embodiments, the detection of the residual amount of dsRNA is performed according to the instruction manual of the dsRNA (modified) quantification detection kit.

[0038] Abbreviations and Terms

[0039] DNA: Deoxyribonucleic acid.

[0040] PCR: polymerase chain reaction.

[0041] mRNA: messenger ribonucleic acid.

[0042] GTP: guanosine triphosphate.

[0043] IVT: in vitro transcription.

[0044] 20x Buffer: 200 mM DTT, 800 mM Tris-HCl, 40 mM spermidine (pH 7.5, 25 °C).

[0045] The term "mRNA" for messenger RNA refers to a polyribonucleotide that encodes at least one polypeptide. The mRNA used herein includes modified and unmodified mRNA.

[0046] The term "mRNA integrity" generally refers to the quality of mRNA, and the mRNA integrity generally refers to the percentage of full-length mRNA transcribed after purification. The mRNA integrity herein is determined by TAE agarose gel electrophoresis.

[0047] The term "capping rate" generally refers to the percentage of capped fragments over the sum of uncapped and capped fragments. The mRNA capping rate herein is determined by LC-MS method.

[0048] Example 1, Cap2-mRNA synthesis kit

[0049] This example provides a Cap2-mRNA synthesis kit, and the product composition of the kit is shown in Table 3.

[0050] Table 3

[0051] Wherein, the structure of Cap2 cap analog Cap2(2-6) is as follows:

[0052] Cap2(2-6) can be synthesized by the following method:

[0053] Synthesis of compound 6A-2:

[0054] At 25±5 °C, 200 g of compound 6A-1 was dissolved in 1200 mL of N,N- dimethylformamide, 192 g of imidazole was added at 25±5 °C, and stirred to dissolve, 254 g of tert-butyldimethylsilyl chloride was slowly added, and the temperature was controlled below 35 °C, after the addition was completed, the reaction was stirred at 25±5 °C for 6 h, and the reaction end point was judged by sampling and monitoring. TLC point plate (thin layer chromatography) showed that compound 6A-1 was completely reacted. 1200 mL of water was added to the above reaction solution to quench the reaction, and 1000 mL of ethyl acetate was added twice to extract, and the organic phase was separated, dried over 1000 mL of saturated brine, and concentrated under reduced pressure to obtain 372 g of yellow oil compound 6A-2, which was directly used in the next step.

[0055] Synthesis of compound 6A:

[0056] At 25±5 °C, 372 g of compound 6A-2 (crude product) was dissolved in 800 mL of tetrahydrofuran and 400 mL of water, stirred to dissolve, and 400 mL of trifluoroacetic acid was added dropwise under ice-salt bath, and the reaction was controlled at 0-3 °C, and stirred for 4 h, and the reaction end point was judged by sampling and monitoring. TLC point plate (thin layer chromatography) showed that compound 6A-2 was completely reacted. 2000 mL of crushed ice was placed in a 5.0 L measuring cup, 215 g of sodium hydroxide was placed on the ice layer, and the reaction solution was slowly poured into the above solution while stirring, and the temperature was controlled below 5 °C, and after the addition was completed, the pH of the solution was adjusted to 8.0-9.0 with saturated sodium bicarbonate, and 2000 mL of ethyl acetate was added twice to extract, and the organic phase was separated, washed with 1000 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 335 g of yellow oil, and 1.5 times the mass of silica gel powder (100-200 mesh) was added to the crude product after concentration, and the sample was stirred, and the silica gel column was purified: mobile phase A: dichloromethane; B: methanol. Gradient (proportion of B phase): 0-10% elution, and the product part was collected, and concentrated to dryness under reduced pressure to obtain 203.4 g of white solid compound 6A.

[0057] 1 H NMR (400 MHz, CDCl3) δ 12.13 (s, 1H), 9.42 (s, 1H), 8.00 (s, 1H), 6.04 (d, J = 16.0 Hz, 1H), 5.32-5.19 (dd, J = 4.0, 4.0 Hz, 1H), 5.03 (s, 1H), 4.57 (m, 1H), 4.12 (m, 1H), 3.75 (m, 1H), 2.78 (m, 1H), 1.24 (d, J = 4.0 Hz, 6H), 0.89 (s, 9H), 0.11 (s, 6H).

[0058] Synthesis of compound 7:

[0059] Take 80 g tetrazole, dissolved in 2800 mL of anhydrous acetonitrile in a three-necked flask, add 97 g of compound 6A at 25±5°C, after stirring and dissolving, replace argon three times, then pour into 200 g of compound 6, the reaction system is stirred at 25±5°C for 2 hours, no obvious exothermic is found, TLC (thin layer chromatography) point plate (DCM:EA:MeOH=10:10:2) shows that the raw material compound 6 is completely reacted, and a new point Int.1 is generated. Then add iodine solution (100 g iodine dissolved in 800 mL of mixed solution THF:H2O:pyridine=8:1:1, prepared into 0.5 mmol / mL iodine solution) dropwise to the solution until the solution no longer fades, a total of 600 mL of iodine solution is added. After the dropwise addition is completed, the reaction solution continues to be stirred for 0.5 hours, sampling is monitored to judge the reaction endpoint. TLC point plate (thin layer chromatography) shows that Int.1 is completely reacted. Add 400-500 mL of saturated sodium thiosulfate solution to the above reaction solution, stir for 15 min, dilute with 1000 mL of water, extract twice with 1000 mL of ethyl acetate, separate the organic phase, wash with 1000 mL of saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 373.2 g of compound 7 as a yellow oily crude product. The crude product is directly used in the next step.

[0060] Synthesis of compound 8:

[0061] At 25±5°C, take 373.2 g of compound 7 crude product, dissolve it in a mixed solution of 1500 mL of glacial acetic acid and 375 mL of water, stir and ultrasonic to fully dissolve, then stir at 25±5°C for 14 hours, sample monitoring to judge the reaction endpoint. TLC point plate (thin layer chromatography) shows that compound 8 is completely reacted. The reaction solution is concentrated under reduced pressure at 40°C, after removing most of the glacial acetic acid, the obtained oily substance is dissolved in 400-500 mL of methanol and concentrated, which is repeated three times to remove the remaining most of the glacial acetic acid, obtaining 400 g of orange-red oily substance. Add silica gel powder (100-200 mesh) with a mass 1.2 times that of the concentrated crude product to the sample, purify by silica gel column, mobile phase A: ethyl acetate; B: methanol diluted with 10 times volume of dichloromethane. Gradient (volume ratio of B phase): 0-100% elution. Collect the product part and concentrate to dryness under reduced pressure to obtain 152 g of yellow solid compound 8.

[0062] Synthesis of compound 9:

[0063] Take 41.4 g tetrazole, dissolved in 1600 mL of anhydrous acetonitrile in a three-necked flask, add 152 g of compound 8 at 25±5°C, replace argon three times after stirring and dissolving, add 130.9 g of compound 8A, after adding, the reaction system is stirred at 25±5°C for 2 hours, no obvious exothermic is found, TLC (thin layer chromatography) (DCM:EA:MeOH=5:5:1) shows that compound 8 is completely reacted, and a new point Int.1 is generated. Then add iodine solution (100 g of iodine dissolved in 800 mL of mixed solution of THF:H2O:pyridine=8:1:1, prepared into 0.5 mmol / mL iodine solution) dropwise to the solution until the solution is no longer faded, a total of 600-700 mL of iodine solution is added. After the dropwise addition is completed, the reaction liquid continues to be stirred for 0.5 hours, and the sample is monitored to judge the reaction endpoint. Standard: TLC (thin layer chromatography) Int.1 reaction is complete. Add 500 mL of saturated sodium thiosulfate solution to the above reaction liquid, stir for 15 min, dilute with 2000 mL of water, extract twice with 1500 mL of ethyl acetate, separate the phases, and dry the obtained organic phase with 1000 mL of saturated sodium chloride solution, then concentrate under reduced pressure to obtain 180 g of yellow oily compound 9 crude product. Add silica gel powder (100-200 mesh) 1.2 times the mass of the crude product after concentration to stir the sample, and purify by silica gel column: mobile phase A: ethyl acetate; B: methanol diluted with 10 volumes of dichloromethane; flow rate: 100 mL / min; gradient (volume ratio of B phase): 0-100% elution. Collect the product part and concentrate to dryness under reduced pressure to obtain 153 g of yellow solid compound 9.

[0064] Synthesis of compound 10:

[0065] Take 50.0 g of compound 9 and dissolve in 1000 mL of methanol at 25±5°C, add 1000 mL of ammonia water at 25±5°C, stir and dissolve, heat to 40°C, stir for 45 h, take sample HPLC to monitor and judge the reaction endpoint. HPLC (peak area) shows that compound 9 is <5.0%, and the remaining amount of intermediate state is <5.0%; the reaction liquid is concentrated under reduced pressure at 40°C, and then dissolved with 300 mL of methanol to concentrate three times to remove most of the remaining ammonia water, to obtain 51.3 g of yellow oily compound 10. The crude product is directly used in the next step.

[0066] Synthesis of compound 11:

[0067] At 25±5℃, 51.3g of compound 10 (crude product) was weighed and dissolved in 60mL of dimethyl sulfoxide. After stirring and dissolving, 60mL of triethylamine trihydrofluoride was added. The reaction was stirred at 40℃ for 2h, and HPLC sampling was used to monitor and determine the reaction endpoint. Compound 10 < 5.0%. The reaction solution was diluted with 6L of water and adjusted to pH 5.3–5.7 with NaOH (1M) solution, with the temperature controlled at 15–20℃ during the addition process. Then, the reaction solution was diluted with pure water until the conductivity was below 10000μs / cm, ready for sample loading and purification. Column equilibration: First, equilibrate the column with 10 L of 2M NaCl at a flow rate of 60 mL / min, then equilibrate with 10 L of pure water at a flow rate of 100 mL / min; Load 7 L of sample treatment buffer at a flow rate of 40 mL / min; Elution: Flow rate of 60 mL / min, gradient of 0–0.25 M NH4HCO3; Start collecting product components when the eluent concentration is 0.20–0.25 M NH4HCO3. Pre-cool the required ultrapure water to 2-8℃; pour the fraction obtained in the previous purification step into a nanofiltration unit for nanofiltration, and monitor the conductivity at the outlet in a timely manner. When the solution is concentrated to 1L, dilute with water and continue nanofiltration. Add 3L of water and nanofiltration for 20 minutes, then concentrate the solution to 1L again. Repeat this operation until the conductivity at the outlet is less than 200μs / cm, at which point concentration is stopped; release approximately 800mL of the solution from the storage tank, then add 800mL × 2 of water to clean the pipeline residue, and finally collect all the fractions; dispense the above solution into six 1L single-necked bottles, pre-freeze in dry ice ethanol solution, and then freeze-dry in a manifold freeze dryer for 40 hours. 25.6g of compound 11 as a white solid was obtained.

[0068] 1 H NMR(400MHz,D2O)δ8.09(s,1H),7.74(s,1H),7.52(s,1H),5.96(d,J=16.0Hz,1H),5.79(d,J=16.0Hz,1H),5.31-5.18(dd,J=4.0,4.0Hz,1 H),5.06-4.92(dd,J=4.0,4.0Hz,1H),4.75-4.64(m,1H),4.52-4.43(m,1H),4.32(d,J=4.0Hz,1H),4.21-4.13(m,3H),4.20-3.97(m,2H).

[0069] Synthesis of compound R17 (i.e., Cap2(2-6), a Cap2 cap analogue):

[0070] Into a 3-necked flask, 10.0 g of compound 11, 15.5 g of compound 1B, 300 mL of DMSO were added under argon protection at 25±5℃, stirred for 10 minutes, the solution was a colorless turbid liquid, 35 g of ZnCl2 was added to the above reaction solution, the temperature was controlled at 15-25℃, after the addition was completed, the reaction was continued to stir at 25±5℃ for 35 hours. HPLC was used to determine the end point of the reaction. The standard compound 11 was less than 20%. 110 g of disodium EDTA dihydrate was weighed into 3.5 L of H2O, stirred and dissolved, then the obtained solution was adjusted to pH = 7.0-7.5 with saturated sodium bicarbonate solution and cooled to 0℃, the reaction solution was poured into the above solution, stirred for 20 min, diluted with pure water to a solution conductivity below 10000 μs / cm, then adjusted to pH = 5.3-5.7 with dilute hydrochloric acid (1M) solution, and the temperature was controlled below 10℃ during the addition. After adjustment, the sample was prepared for purification. Column equilibration: first equilibrate the column with 3L of 2M NaCl at a flow rate of 60 mL / min, then equilibrate with pure water at a flow rate of 60 mL / min for 4L; sample treatment solution 5L at a flow rate of 50 mL / min; elution: flow rate 50 mL / min, gradient 0-0.10M NH4HCO3; when the eluent contains 0.05-0.10M NH4HCO3, start collecting the product component. The obtained product purity is greater than 95%, the components are collected at 2-8℃, diluted with pure water to a solution conductivity of 10000 μs / cm, adjusted to pH = 5.3-5.7, purified twice, the obtained product purity is greater than 95%, the components are collected and stored at 2-8℃, the required ultra-pure water is pre-cooled to 2-8℃; the two batches of components obtained in the purification are poured into a nanofiltration machine for nanofiltration operation, and the pH of the components and the conductivity of the outlet are detected in time. When the solution is concentrated to 1L, start diluting with water and continue nanofiltration, add 3L of water, nanofiltration for 20 min, continue to concentrate the solution to 1L, repeat the operation, finally the conductivity of the outlet is less than 200 μs / cm, stop concentrating; pour out about 800 mL of the solution components in the storage tank, add 800 mL of water twice to wash the residual in the pipeline, finally collect all the components; divide the above solution into 6 1L single-necked flasks, pre-freeze in dry ice ethanol solution, then connect to a multi-way freeze dryer for freeze-drying for 40 hours. Freeze-drying 6.85 g of white solid R17 is obtained.

[0071] 1H NMR (400 MHz, D20) δ 9.11 (s, 1H), 8.31 (s, 1H), 8.07 (s, 1H), 7.77 (s, 1H), 6.17 (d, J = 16.0 Hz, 1H), 6.07 (d, J = 20.0 Hz, 1H), 5.85 (d, J = 4.0 Hz, 1H), 5.45-5.37 (dd, J = 4.0, 4.0 Hz, 1H), 5.32-5.24 (d, J = 4.0, 4.0 Hz, 1H), 4.94 (m, 1H), 4.72 (m, 2H), 4.13-4.52 (m, 9H), 4.04 (s, 3H), 3.48 (s, 3H)

[0072] 31 P NMR (162 MHz, D20) δ -1.38 (s, 1H), -11.56 (t, J = 13.8 Hz, 2H), -22.82 (t, J = 17.8 Hz, 1H).

[0073] Cap2-mRNA synthesis kit of the present embodiment was used to prepare capped mRNA by in vitro transcription experiment. The experimental parameters are shown in Table 4.

[0074] Table 4

[0075] During the experiment, first calculate the volume of the required materials in the system, then add the sample. First add Distilled water distilled water in the system, then add NTPs, 20x IVT Reaction Buffer, magnesium chloride solution, cap analogs in turn, mix well and centrifuge gently, then add nuclease inhibitor, inorganic pyrophosphatase, T7 RNA polymerase, DNA template (coding EGFP gene), mix well and centrifuge gently, add DNase I 2U at 42.7°C for 3.25h, continue to incubate at 38°C for 30min, then purify the mRNA with lithium chloride solution. The agarose gel electrophoresis detection diagram of the purified mRNA is shown in Figure 1.

[0076] Example 2, cap analog co-transcription capping experiment of different concentrations of magnesium chloride solution

[0077] EGFP was used as the DNA template for in vitro transcription.

[0078] Prepare the in vitro transcription reaction system: the reaction system is 20μl, and the specific components are shown in Table 5.

[0079] Table 5

[0080] The magnesium chloride solution of each experimental group was 10mM, 15mM, 20mM, 30mM, 40mM respectively.

[0081] 37℃ reaction for 4h, then recover mRNA product with lithium chloride solution; use dsRNA quantitative detection kit to detect residual amount of dsRNA.

[0082] Results are shown in Table 6.

[0083] Table 6

[0084] Results show that in the IVT reaction system of the present application, the preferred concentration of magnesium chloride solution is 15mM-40mM, more preferably 20mM-30mM.

[0085] Example 3, co-transcriptional capping experiment of cap analogs with different concentrations

[0086] Experimental method is the same as Example 2, magnesium chloride solution is 20mM, and cap analogs are 1.0mM, 2.5mM, 5.0mM, 6.5mM, 7.0mM, 7.5mM, 8.0mM, and 10.0mM, respectively.

[0087] Results are shown in Table 7.

[0088] Table 7

[0089] Results show that when the final concentration of cap analog is 1.5mM, the capping rate is ≥98%, and there is no significant difference between the yields. In the IVT reaction system of the present application, the final concentration of cap analog is ≥1.5mM, preferably 1.5mM-10mM, more preferably 2.5mM-10mM.

[0090] Example 4, co-transcriptional capping experiment of cap analogs with different transcription temperatures

[0091] Experimental method is the same as Example 2, and the concentration of cap analog is 5mM. Reaction temperatures are 36.0℃, 37.0℃, 37.5℃, 38.0℃, 38.7℃, 39.3℃, 40.2℃, 41.2℃, 42.0℃, 42.7℃, and 43.5℃, respectively.

[0092] Results are shown in Table 8 and Table 9:

[0093] Table 8

[0094] Table 9

[0095] Results show that as the reaction temperature increases, the mRNA yield also increases, and the residual amount of dsRNA is ≤0.1%. In the IVT reaction system of the present application, the transcription temperature is 36.0℃-43.5℃, preferably 40.0℃-43.5℃, more preferably 42.0℃-42.7℃.

[0096] Example 5, Vaccines of Different Cap Analog

[0097]

[0098] The specific structure of each cap is shown in Table 10. In addition to being commercially available, each cap analog can also be synthesized according to the method described in CN202310391954.9 and / or CN118373866A (CN202310091020.3), the entire contents of which are hereby incorporated by reference.

[0099] Table 10, Different cap analog names and structures

[0100] Using T19 (9-heptadecyl octanoate 8-((7-((3-(hexylthio)-2-methylpropanoyl)oxy)n- heptyl)(2-hydroxyethyl)amine): Cholesterol: DSPC: PEG 2k -DMG = 50:38.5:10:1.5 ratio dissolved in anhydrous ethanol to form organic phase A (the average concentration of lipids is 10 mg / ml), select the mRNA transcribed from SEQ ID NO: 1 (modified with different cap analogs in the in vitro transcription process, and modified with 100% m1ψ, with the same 5'UTR, 3'UTR, 3'PolyA tail), dissolved in pH = 4.0 citric acid / sodium citrate buffer to form aqueous phase B (mRNA concentration is 0.54 mg / ml), use a microfluidic device to mix solution A: solution B at a volume ratio of 1:3 to prepare mRNA-LNP, after preparation, dialysis in 20 mM Tris buffer for 24 h, and change the solution every 8 h. After 24 h, pass through a 0.22 μm filter to sterilize to obtain mRNA-LNP. C57 mice (10 mice / group, female, priming age 8 weeks, body weight 18-22 g) were set up as experimental groups and blank control groups, the experimental groups were separated by 2 weeks, and the C57 mice were immunized with LNP containing 5 μg of the above different mRNA by intramuscular injection twice, 2 weeks after each immunization, the experimental and control mice were taken blood from the eye socket and centrifuged to obtain serum, which was stored at -20 degrees Celsius for later use; 2 weeks after the second immunization, the spleens of the experimental and control groups were removed for later use.

[0101] The serum of the experimental group mice was diluted by 10000x, 20000x, 40000x, 80000x, 160000x, 320000x, 640000x, and was added to the ELISA plate coated with the standard gE protein, 100 μL per sample, 3 parallel wells, 4°C incubation for 2h. The 96-well plate after incubation with the first antibody was taken out, and the liquid was poured out and dried on the absorbent paper until there was no residue in the hole. The diluted secondary antibody (Goat pab to Ms IgG (HRP), 1:50000) was added to the 96-well plate at a volume of 100 μL per well, and the plate was incubated at room temperature for 1h. The enzyme-labeled plate after incubation with the secondary antibody was taken out, and the liquid was poured out and dried on the absorbent paper until there was no residue in the hole. The TMB (3,3',5,5'-Tetramethylbenzidine) solution was poured into the sample slot, 100 μL of TMB was added to each well with the gun, and the color was developed at room temperature for 20 minutes. At this time, the positive sample showed blue. The sample should record the liquid addition and color development reaction time of each plate to ensure consistent incubation time. After color development, 100 μL of stop solution was added to each well with the gun. At this time, the sample showed yellow, and the absorbance was measured at 450nm. After adding the stop solution to each plate, the plate was read within two minutes.

[0102] In a six-well plate, add mouse lymphocyte separation medium, put into 70 μm cell screen, grind the spleen of the immunized mouse into colorless with the syringe piston, suck the lower cell suspension into a 15ml centrifuge tube, slowly add 1ml RPMI1640 medium to the cell suspension layer (do not break the interface), 800g, 30min room temperature centrifugation (rise 3 fall 3), suck the lymphocyte layer, add 10mL RPMI-1640, 350g, 10min room temperature centrifugation, discard the supernatant, 2mL RPMI-1640 complete medium (+10% inactivated serum +1% double antibody) resuspension to prepare spleen cell solution. Add the spleen cells to the cell culture 2x10 6Individual wells were stimulated with corresponding peptide library (2 pg / mL) or equal volume of solvent, and incubated at 37 °C for 1 h. Then, 0.4 pL of protein transport inhibitor was added to each well, and incubated at 37 °C for 5 h. After that, the cells in the wells were collected into 2 mL centrifuge tubes, centrifuged at 350 g at 4 °C for 5 min, and the supernatant was discarded. Then, 100 pL of Zombie staining solution was added to each well, and centrifuged to discard the supernatant. Subsequently, 1% BSA containing PBS solution with anti-mouse CD16 / CD32 (Biolegend) mAb, 50 pL of a mixture of anti-mouse CD3 (Biolegend), anti-mouse CD4 (Biolegend), and anti-mouse CD8 (Biolegend) mAbs, 150 pL of Fixation / Permeabilization solution (Biolegend), and a mixture of anti-mouse IFN-g (Biolegend) and anti-mouse IL-2 (Biolegend) were sequentially added to each well. After each step, the supernatant was discarded by centrifugation. Finally, 200 pL of 1% BSA containing PBS was added to each well, and the cells were detected by flow cytometry. The proportions of CD3 + CD4 + IFN-g + , CD3 + CD8 + IFN-g + , CD3 + CD4 + IL-2 + , CD3 + CD8 + IL-2 + cells stimulated with and without polypeptides were recorded, and the data were processed by CytExpert 2.4 software.

[0103] The results are shown in FIG. 2 and Tables 11 and 12.

[0104] Table 11. Antibody expression levels in different cap mRNA experiments

[0105] Table 12. IL2 levels in different cap mRNA experiments + and IFN-g + CD4 + T cell factor levels

[0106] The above embodiments are only used for illustrating the present application, and are not intended to limit the present application. Those skilled in the art can make various changes and modifications to achieve the same purpose without departing from the spirit and scope of the present application. Therefore, all equivalent technical solutions also belong to the protection scope of the present application.

Claims

1. An in vitro co-transcriptional RNA capping reagent composition comprising: Cap2 cap analog, magnesium chloride and DNA template.

2. The composition of claim 1, wherein, The Cap2 cap analog has the following structure of Formula I: In formula I: R1is selected from -H, -OH, C 1-6 alkyl and C 1-6 alkoxy; R2is selected from -H, -OH, halo, C 1-6 any one of -OH, -OCH3, halo, C R3, R0 are each independently selected from any one of F, Cl, Br, I; J1, J2 are each independently selected from natural or modified pyrimidine nucleotide base, natural or modified purine nucleotide base.

3. The composition of claim 1, wherein, The Cap2 cap analog is selected from one or more of the following compounds:

4. The composition according to any one of claims 1-3, further comprising one or more of nucleotide triphosphate molecules, RNA polymerase; Preferably, the composition further comprises one or more of RNAse inhibitor, inorganic pyrophosphatase, buffer; Preferably, the composition further comprises one or more of DNAse, LiCl precipitation solution.

5. An in vitro co-transcriptional RNA capping kit comprising the composition according to any one of claims 1-4.

6. Use of the composition according to any one of claims 1-4 or the kit according to claim 5 in the preparation of 5' capped RNA.

7. A method for in vitro co-transcriptional RNA capping, the method comprising performing template transcription in a transcription reaction system comprising the composition according to any one of claims 1-4 to synthesize 5' capped RNA.

8. The method of claim 7, wherein, The concentration of magnesium chloride in the transcription reaction system is 15mM-40mM, preferably 20mM-30mM.

9. The method of claim 7, wherein, The concentration of Cap2 cap analog in the transcription reaction system is ≥1.5mM, preferably the concentration is 1.5mM-10mM, more preferably 2.5mM-10mM.

10. The method of claim 7, wherein, The transcription reaction temperature is 36.0℃-43.5℃, preferably 40.0℃-43.5℃, more preferably 42.0℃-42.7℃.

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