Cap2-mRNA synthesis reagent composition, kit and application of Cap2-mRNA synthesis reagent composition

Abstract

The invention provides a Cap2-mRNA synthesis reagent composition, a kit and application of the Cap2-mRNA synthesis reagent composition and the kit. Specifically, the invention provides an in-vitro co-transcription RNA capping reagent composition, which comprises a Cap2 cap analogue, magnesium chloride and a DNA template. According to the invention, the Cap2 cap analogue is used and does not form competitive inhibition with GTP, so that the utilization rate of the cap analogue is improved, and the mRNA synthesized by transcription reaction is high in capping efficiency, high in yield and high in integrity.

Description

Technical Field

[0001] This invention relates to a Cap2-mRNA synthesis reagent composition, kit, and its application. Background Technology

[0002] In vitro transcriptional synthesis of mRNA involves using plasmid DNA as a template in a cell-free system to transcribe mRNA using RNA polymerase, and then adding modifications such as a 5' cap and a 3' poly(A) tail to mimic the in vivo mRNA synthesis process. The mRNA capping process is a crucial step in in vitro transcriptional synthesis. The mRNA cap structure can be achieved through enzymatic modification using capping enzymes and 2-O-methyltransferases after transcription, or through co-transcriptional capping with cap analogs.

[0003] In the co-transcriptional capping method, the cap analog and GTP compete for the mRNA initiation sequence. Therefore, the molar ratio of cap analog to GTP is an important factor affecting the capping rate in co-transcriptional capping of mRNA.

[0004] Currently, the most commonly used cap analogues are the Cap1 structure. However, the nucleotides of Cap1 cap analogues have multiple active groups, requiring multiple protection and deprotection reactions during synthesis, resulting in low mRNA synthesis efficiency. Summary of the Invention

[0005] One object of the present invention is to provide an improved in vitro transcription reaction system containing a structural cap analogue.

[0006] Another object of the present invention is to provide a method for preparing mRNA using Cap2 structural cap analogs.

[0007] Another object of the present invention is to provide a kit for preparing 5' capped mRNA using Cap2 structural cap analogs.

[0008] On one hand, the present invention provides an in vitro co-transcription RNA capping reagent composition comprising: a Cap2 cap analog, magnesium chloride, and a DNA template.

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

[0010] According to a specific embodiment of the present invention, in the in vitro co-transcriptional RNA capping reagent composition of the present invention, the Cap2 cap analog has the structure shown in Formula I:

[0011]

[0012] In Formula I:

[0013] R1 is selected from -H, -OH, C. 1-6 Alkyl and C 1-6 Any one of the alkoxy groups;

[0014] R2 is selected from -H, -OH, halogens, and C. 1-6 Any one of the alkoxy groups;

[0015] R3 and R0 are each independently selected from any one of F, Cl, Br, and I;

[0016] J1 and J2 are each independently selected from natural or modified pyrimidine nucleotide bases and natural or modified purine nucleotide bases.

[0017] According to a specific embodiment of the present invention, in the in vitro co-transcriptional RNA capping reagent composition of the present invention, the Cap2 cap analogue is selected from one or more of the following compounds:

[0018]

[0019]

[0020]

[0021]

[0022]

[0023] According to a specific embodiment of the present invention, in the in vitro co-transcriptional RNA capping reagent composition of the present invention, the Cap2 structural cap analog can be synthesized according to the method described in CN202310391954.9, the entire contents of which are incorporated herein by reference.

[0024] According to a specific embodiment of the present invention, the in vitro co-transcriptional RNA capping reagent composition of the present invention may further include one or more common in vitro co-transcriptional RNA capping reagents such as nucleotide triphosphate molecules and RNA polymerase. Preferably, the in vitro co-transcriptional RNA capping reagent composition of the present invention further includes one or more of RNase inhibitors, inorganic pyrophosphatase, and buffer solutions. Preferably, the in vitro co-transcriptional RNA capping reagent composition of the present invention may further include one or more of DNase and LiCl precipitation solution. The specific selection and dosage of the nucleotide triphosphate molecules, RNA polymerase, RNase inhibitors, inorganic pyrophosphatase, buffer solutions, DNase, and LiCl precipitation solution can be determined according to the commonly used reagents and dosages in the in vitro co-transcriptional RNA capping process in the prior art.

[0025] On the other hand, the present invention also provides an in vitro co-transcription RNA capping kit, which includes the aforementioned in vitro co-transcription RNA capping reagent composition of the present invention.

[0026] On the other hand, the present invention 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.

[0027] On the other hand, the present invention also provides a method for in vitro co-transcriptional RNA capping, the method comprising performing template transcription in a transcription reaction system containing the in vitro co-transcriptional RNA capping reagent composition described in the present invention to synthesize 5' capped RNA.

[0028] According to a specific embodiment of the present invention, the RNA is mRNA.

[0029] According to a specific embodiment of the present invention, in the in vitro co-transcription RNA capping method of the present invention, the concentration of magnesium chloride in the transcription reaction system is 15mM to 40mM, preferably 20mM to 30mM.

[0030] According to a specific embodiment of the present invention, in the in vitro co-transcription of RNA capping method of the present invention, the concentration of Cap2 cap analog in the transcription reaction system is ≥1.5mM, preferably 1.5mM to 10mM, more preferably 2.5mM to 10mM.

[0031] According to a specific embodiment of the present invention, in the in vitro co-transcription of RNA capping method of the present invention, the transcription reaction temperature is 36.0℃-43.5℃, preferably 40.0℃-43.5℃, and more preferably 42.0℃-42.7℃.

[0032] This invention uses a Cap2 analogue, which does not competitively inhibit GTP, thus improving the utilization rate of the cap analogue. The resulting mRNA synthesized via transcription has high capping efficiency, high yield, and high integrity. A complete in vitro transcription reaction system is provided. This reduces the amount of cap analogue required, thereby lowering material costs in the capped mRNA production process. Attached Figure Description

[0033] Figure 1 This is an agarose gel electrophoresis image of the capped mRNA prepared in Example 1.

[0034] Figure 2 This study demonstrates the effect of mRNA-LNPs formed from mRNAs with different capping methods on the expression levels of gE-specific binding antibodies and CD4+ cytokines in cap analog experiments. Detailed Implementation

[0035] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0036] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and reagents used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, equipment, and materials similar to or equivalent to those described in the embodiments of this invention may be used to implement this invention.

[0037] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0038] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0039] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment / implementation, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0040] In each embodiment, the general instrument and reagent information is shown in Table 1 and Table 2.

[0041] Table 1. Instrument Information

[0042]

[0043]

[0044] Table 2. Material Source Information

[0045]

[0046] In each embodiment, the detection of residual dsRNA was performed in accordance with the instructions of the dsRNA (modified) quantitative detection kit.

[0047] Abbreviations and terms

[0048] DNA: deoxyribonucleic acid.

[0049] PCR: Polymerase chain reaction.

[0050] mRNA: messenger ribonucleic acid.

[0051] GTP: Guanosine triphosphate.

[0052] IVT: In vitro transcription.

[0053] 20×Buffer: 200mM DTT, 800mM Tris-HCl, 40mM spermidine (pH7.5, 25°C).

[0054] The term "mRNA" stands for messenger RNA, which refers to a polynucleotide that encodes at least one polypeptide. The mRNAs used in this article include both modified and unmodified mRNAs.

[0055] The term "mRNA integrity" usually refers to the quality of mRNA, which generally refers to the percentage of full-length mRNA transcribed after purification. In this article, mRNA integrity was determined using TAE agarose gel electrophoresis.

[0056] The term "capping rate" generally refers to the percentage of capped fragments out of the sum of uncapped and capped fragments. In this article, the mRNA capping rate was determined using LC-MS.

[0057] Example 1: Cap2-mRNA Synthesis Kit

[0058] This embodiment provides a Cap2-mRNA synthesis kit, the composition of which is shown in Table 3.

[0059] Table 3

[0060] Component Name concentration Specification Item number <![CDATA[H2O(RNase-free)]]> NA 1ml NA ATP 100mM 30μl NA CTP 100mM 30μl NA N1-Me-Pseudo-UTP 100mM 30μl EM2001 UTP 100mM 30μl EM2002 GTP 100mM 30μl NA DNase I 1U / μl 40μl EM4003 Cap2(2-6) 100mM 20μl EM1003 IVT Reaction Buffer 20× 20μl NA T7 RNA polymerase Mix NA 180μl NA LiCl precipitate solution 7.5M 1ml NA DNA template NA 20μl NA magnesium chloride solution 1M 20μl NA manual NA 1 copy NA

[0061] The structure of the Cap2 analogue Cap2(2-6) is as follows:

[0062]

[0063] Cap2(2-6) can be synthesized as follows:

[0064]

[0065] Synthesis of compound 6A-2:

[0066] At 25±5℃, 200g of compound 6A-1 was weighed and dissolved in 1200mL of N,N-dimethylformamide. 192g of imidazole was added at 25±5℃, and the mixture was stirred until dissolved. 254g of tert-butyldimethylchlorosilane was slowly added, maintaining the temperature below 35℃. After the addition was complete, the mixture was stirred at 25±5℃ for 6 hours. Samples were taken for monitoring to determine the reaction endpoint. TLC (thin-layer chromatography) showed that compound 6A-1 had reacted completely. 1200mL of water was added to the above reaction solution to quench the reaction. Extraction was performed twice with 1000mL of ethyl acetate. The resulting organic phase was separated, washed with 1000mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 372g of a pale yellow oily compound 6A-2. The crude product was directly added to the next step.

[0067] Synthesis of compound 6A:

[0068] At 25±5℃, 372g of compound 6A-2 (crude product) was weighed and dissolved in 800mL of tetrahydrofuran and 400mL of water. The mixture was stirred until dissolved, and 400mL of trifluoroacetic acid was added dropwise under an ice-salt bath. The reaction temperature was controlled between 0-3℃, and the mixture was stirred for 4 hours. Samples were taken for monitoring, and the reaction endpoint was determined. TLC (thin-layer chromatography) showed that compound 6A-2 had reacted completely. 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. While stirring, the reaction solution was slowly poured into the above solution, maintaining the temperature below 5 °C. After the addition was complete, the pH of the solution was adjusted to between 8.0 and 9.0 with saturated sodium bicarbonate. Extraction was performed twice with 2000 mL of ethyl acetate. The liquid was separated, and the resulting organic phase was washed with 1000 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 335 g of a yellow oily substance. Silica gel powder (100-200 mesh) was added at 1.5 times the weight of the concentrated crude product and mixed. Silica gel column purification was performed: mobile phase A: dichloromethane; B: methanol. Gradient (proportion of phase B): 0–10% elution. The product fraction was collected and concentrated to dryness under reduced pressure to obtain 203.4 g of a white solid compound 6A.

[0069] 1H NMR(400MHz, CDCl3)δ12.13(s,,1H),9.42(s,1H),8.00(s,1H),6.04(dd,1H),5.32(dd,1H), 4.57(m,1H),4.12(m,1H),3.75(m,1H),2.78(m,1H),1.24(d,6H),0.89(s,9H),0.11(s,6H).

[0070] Synthesis of compound 7:

[0071] Weigh 80g of tetrazolium and dissolve it in 2800mL of anhydrous acetonitrile in a three-necked flask. Add 97g of compound 6A at 25±5℃. After stirring and dissolving, purge with argon three times. Then add 200g of compound 6. Stir the reaction system at 25±5℃ for 2 hours. No significant exothermic reaction was observed. TLC (thin-layer chromatography) spotting (DCM:EA:MeOH = 10:10:2) showed that the starting material compound 6 reacted completely, with a new spot Int.1 appearing. Then, add iodine solution dropwise (100g of iodine dissolved in 800mL of a mixed solution of THF:H2O:pyridine = 8:1:1, to prepare a 0.5mmol / mL iodine solution) until the solution no longer decolorizes. A total of 600mL of iodine solution was added. After the addition was complete, the reaction solution was stirred for 0.5 hours. Samples were taken for monitoring to determine the reaction endpoint. TLC (thin-layer chromatography) showed that the reaction att1 was complete. 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 layers, wash the organic phase with 1000 mL of saturated brine, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and obtain 373.2 g of compound 7 as a yellow oily crude product. The crude product was directly used in the next step.

[0072] Synthesis of compound 8:

[0073] At 25±5℃, 373.2g of crude compound 7 was weighed and dissolved in a mixed solution of 1500mL glacial acetic acid and 375mL water. The solution was stirred and sonicated until fully dissolved, then stirred at 25±5℃ for 14 hours. Samples were taken for monitoring to determine the reaction endpoint. TLC (thin-layer chromatography) showed that compound 8 reacted completely. The reaction solution was concentrated under reduced pressure at 40℃ to remove most of the glacial acetic acid. The resulting oily substance was dissolved in 400-500mL methanol and concentrated. This cycle was repeated three times to remove most of the remaining glacial acetic acid, yielding 400g of an orange-red oily substance. Silica gel powder (100-200 mesh) of 1.2 times the weight of the concentrated crude product was added and mixed. The mixture was purified by silica gel column chromatography. Mobile phase A: ethyl acetate; B: methanol diluted with ten times the volume of dichloromethane. Gradient (volume ratio of phase B): 0-100% elution. The product fraction was collected and concentrated to dryness under reduced pressure to obtain 152g of yellow solid compound 8.

[0074] Synthesis of compound 9:

[0075] Weigh 41.4 g of tetrazolium and dissolve it in 1600 mL of anhydrous acetonitrile in a three-necked flask. Add 152 g of compound 8 at 25 ± 5 °C. After stirring and dissolving, purge with argon three times, then add 130.9 g of compound 8A. After the addition is complete, stir the reaction system at 25 ± 5 °C for 2 hours. No significant exothermic reaction was observed. TLC (thin-layer chromatography) (DCM:EA:MeOH = 5:5:1) showed that compound 8 reacted completely, with a new spot Int.1 appearing. Then, add iodine solution dropwise (100 g of iodine dissolved in 800 mL of a mixed solution of THF:H2O:pyridine = 8:1:1, to prepare a 0.5 mmol / mL iodine solution) until the solution no longer decolorizes. A total of 600–700 mL of iodine solution was added. After the addition was complete, the reaction solution was stirred for 0.5 hours. Samples were taken for monitoring to determine the reaction endpoint. Standard: TLC (thin-layer chromatography) Int.1 indicates complete reaction. Add 500 mL of saturated sodium thiosulfate solution to the above reaction solution, stir for 15 min, dilute with 2000 mL of water, extract twice with 1500 mL of ethyl acetate, separate the liquid and liquid phases, wash the obtained organic phase with 1000 mL of saturated sodium chloride solution, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 180 g of crude yellow oily compound 9. Add 1.2 times the mass of the concentrated crude product of silica gel powder (100-200 mesh) and mix. Purify with silica gel column: mobile phase A: ethyl acetate; B: methanol diluted with ten times the volume of dichloromethane; flow rate: 100 mL / min; gradient (volume ratio of phase B): 0-100% elution. Collect the product fraction, concentrate to dryness under reduced pressure to obtain 153 g of yellow solid compound 9.

[0076] Synthesis of compound 10:

[0077] At 25±5℃, 50.0 g of compound 9 was weighed and dissolved in 1000 mL of methanol. Then, 1000 mL of ammonia was added at 25±5℃, and the mixture was stirred until dissolved. The mixture was heated to 40℃ and stirred for 45 h. HPLC monitoring was performed to determine the reaction endpoint. HPLC (peak area) showed that compound 9 < 5.0%, and the remaining intermediate amount < 5.0%. The reaction solution was concentrated under reduced pressure at 40℃, then dissolved and concentrated three times with 300 mL of methanol to remove most of the remaining ammonia, yielding 51.3 g of a pale yellow oily compound 10. The crude product was directly added to the next step.

[0078] Synthesis of compound 11:

[0079] 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 samples were taken for monitoring to 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 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 continue to concentrate the solution to 1L. 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 x 2 of water to clean the pipeline residue, and finally collect all the fraction; dispense the above solution into six 1L single-necked flasks, 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 is obtained.

[0080] 1H NMR(400MHz,D2O)δ8.09(s,1H),7.74(s,1H),7.52(s,1H),5.98(d,J=15.4Hz, 1H),5.81(d,J=15.5Hz,1H),5.31(d,J=3.8Hz,1H),5.19(d,J=3.9Hz,1H),5.05 (d,J=4.2Hz,1H),4.92(d,J=4.3Hz,1H),4.80–4.69(m,1H),4.48(ddd,J=21.4 ,7.9,4.4Hz,1H),4.32(d,J=7.0Hz,1H),4.25–4.08(m,3H),4.05–3.91(m,2H).

[0081] Synthesis of compound R17:

[0082] Under argon protection at 25±5℃, 10.0g of compound 11, 15.5g of compound 1B, and 300mL of DMSO were added sequentially to a three-necked flask. The mixture was stirred for ten minutes until a colorless turbid solution was obtained. Then, 35g of ZnCl2 was added to the reaction solution, maintaining the temperature at 15-25℃. After the addition was complete, the reaction was continued at 25±5℃ with stirring for 35 hours. The reaction endpoint was determined. Standard compound 11 < 20%. 110g of EDTA disodium dihydrate was weighed and added to 3.5L of H2O and stirred until dissolved. The resulting 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 and stirred for 20 minutes. The solution was diluted with pure water until the conductivity was below 10000µs / cm. Then, the pH was adjusted to between 5.3-5.7 with dilute hydrochloric acid (1M) solution, maintaining the temperature below 10℃ during the addition process. After adjustment, the solution was ready for purification. Column equilibration: First, equilibrate the column with 3L of 2M NaCl at a flow rate of 60mL / min, then equilibrate with 4L of pure water at a flow rate of 60mL / min; Load 5L of sample treatment buffer at a flow rate of 50mL / min; Elution: Flow rate of 50mL / min, gradient of 0~0.10M NH4HCO3; When the eluent concentration is 0.05~0.10M NH4HCO3, start collecting product components. The fraction with a purity greater than 95% was collected and stored at 2-8℃. It was diluted with pure water until the solution conductivity was 10000 μS / cm, and the pH was adjusted to between 5.3 and 5.7. A second purification was performed, and the fraction with a purity greater than 95% was collected and stored at 2-8℃. The required ultrapure water was pre-cooled to 2-8℃. Two batches of the purified fraction were poured into a nanofiltration unit for nanofiltration, and the pH and conductivity of the outlet were monitored regularly. When the solution was concentrated to 1L, water dilution was added, and nanofiltration continued. 3L of water was added, and nanofiltration was performed for 20 minutes. The solution was then concentrated to 1L, and this process was repeated. Concentration was stopped when the conductivity at the outlet was measured to be less than 200 μS / cm. Approximately 800mL of the solution was discharged from the storage tank, and then 800mL of water was added. After cleaning the pipeline for residue, all components were collected. The solution was dispensed into six 1L single-necked flasks, pre-frozen in dry ice and ethanol solution, and then freeze-dried in a manifold freeze dryer for 40 hours. 6.85g of white solid R17 was obtained after freeze-drying.

[0083] 1H NMR(400MHz,D2O)δ9.11(s,1H),8.31(s,1H),8.07(s,1H),7.77(s,1H),6.17(d,J=1.6,1H),6.07(d,J=2.0Hz,1H),5.85(d,J=4 .0Hz,1H),5.45(d,J=3.2Hz,1H),5.32(d,J=3.2Hz,1H),4.94(m,1H),4.72(m,2H),4.13-4.52(m,9H),4.04(s,3H),3.48(s,3H).

[0084] 31P NMR(400MHz,D2O)δ,-1.38(s,1H),-11.56(m,2H),-22.82(t,,1H).

[0085] Capped mRNA was prepared using the Cap2-mRNA synthesis kit described in this embodiment for in vitro transcription experiments. The experimental parameters are shown in Table 4.

[0086] Table 4

[0087]

[0088] In the experiment, the required volume of materials for the system was first calculated, and then samples were added. First, distilled water was added to the system, followed by NTPs, 20×IVT Reaction Buffer, magnesium chloride solution, and a capping analogue. After mixing, the mixture was gently centrifuged. Then, nuclease inhibitors, inorganic pyrophosphatase, T7 RNA polymerase, and DNA template (encoding the EGFP gene) were added. After thorough mixing, the mixture was gently centrifuged and incubated at 42.7℃ for 3.25 h. Then, 2 U of DNase I was added, and the mixture was incubated at 38℃ for another 30 min. Finally, the mRNA was purified using lithium chloride solution. The agarose gel electrophoresis image of the purified mRNA is shown below. Figure 1 As shown.

[0089] Example 2: Capping experiment of cap analogue co-transcriptionalization with magnesium chloride solutions of different concentrations

[0090] EGFP was used as a DNA template for in vitro transcription.

[0091] Preparation of in vitro transcription reaction system: 20 μl of reaction system, the specific components are shown in Table 5.

[0092] Table 5

[0093]

[0094]

[0095] The magnesium chloride solutions for each experimental group were 10 mM, 15 mM, 20 mM, 30 mM, and 40 mM, respectively.

[0096] After reacting at 37℃ for 4 hours, the mRNA product was recovered with lithium chloride solution; the residual amount of dsRNA was detected using a dsRNA quantification kit.

[0097] The results are shown in Table 6.

[0098] Table 6

[0099] Group Yield (μg) dsRNA residual amount 10mM magnesium chloride solution 26 0.002％ 15mM magnesium chloride solution 77 0.008％ 20mM magnesium chloride solution 111 0.014％ 30mM magnesium chloride solution 103 0.014％ 40mM magnesium chloride solution 90 0.008％

[0100] The results show that in the IVT reaction system of the present invention, the preferred concentration of magnesium chloride solution is 15mM to 40mM, more preferably 20mM to 30mM.

[0101] Example 3: Co-transcriptional capping experiment with different concentrations of cap analogues

[0102] The experimental method was the same as in Example 2, with magnesium chloride solution at 20 mM and cap analogues at concentrations of 1.0 mM, 2.5 mM, 5.0 mM, 6.5 mM, 7.0 mM, 7.5 mM, 8.0 mM, and 10.0 mM.

[0103] The results are shown in Table 7.

[0104] Table 7

[0105]

[0106]

[0107] The results showed that the capping rate was ≥98% when the final concentration of the capping analog was 1.5 mM, and there was no significant difference in yield between different groups. In the IVT reaction system of the present invention, the final concentration of the capping analog is ≥1.5 mM, preferably 1.5 mM to 10 mM, and more preferably 2.5 mM to 10 mM.

[0108] Example 4: Capping Experiment with Cap Analog Co-transcription at Different Transcription Temperatures

[0109] The experimental method was the same as in Example 2, with the cap analog concentration at 5 mM. The reaction temperatures were 36.0℃, 37.0℃, 37.5℃, 38.0℃, 38.7℃, 39.3℃, 40.2℃, 41.2℃, 42.0℃, 42.7℃, and 43.5℃, respectively.

[0110] The results are shown in Tables 8 and 9:

[0111] Table 8

[0112] Group Yield (μg) 37.0℃ 70 42.7℃ 97

[0113] Table 9

[0114]

[0115]

[0116] The results showed that as the reaction temperature increased, the mRNA yield also increased, and the residual dsRNA level was ≤0.1%. The IVT reaction system of this invention has a transcription temperature of 36.0℃-43.5℃, preferably 40.0℃-43.5℃, and more preferably 42.0℃-42.7℃.

[0117] Example 5: Vaccines with different cap analogues

[0118]

[0119] The specific structure of each hat is shown in Table 10. In addition to being obtained commercially, each hat analogue can also be synthesized by referring to the methods described in CN202310391954.9 and / or CN118373866A (CN202310091020.3), the entire contents of which are incorporated herein by reference.

[0120] Table 10. Names and structures of different hat-like items

[0121]

[0122]

[0123] Using T19(8-((7-((3-(hexylthio)-2-methylpropionyl)oxo)n-heptyl)(2-hydroxyethyl)amine)octanoic acid 9-heptadecyl ester):cholesterol:DSPC:PEG 2k DMG was dissolved in anhydrous ethanol at a ratio of 50:38.5:10:1.5 to prepare organic phase A (average lipid concentration of 10 mg / ml). mRNA transcribed from SEQ ID NO:1 (modified with different cap analogs from Table 10 during in vitro transcription, and 100% m1ψ modified, with the same 5'UTR, 3'UTR, and 3'PolyA tail) was dissolved in citrate / sodium citrate buffer at pH 4.0 to prepare aqueous phase B (mRNA concentration of 0.54 mg / ml). Using a microfluidic device, the solution was mixed at a volume ratio of 1:3 (solution A:solution B) to prepare mRNA-LNP. After preparation, the solution was dialyzed against 20 mM Tris buffer for 24 h, with the buffer changed every 8 h. After 24 h, the solution was sterilized by filtering through a 0.22 μm filter to obtain mRNA-LNP. C57 mice (10 mice / group, female, 8 weeks old at initial immunization, weighing 18-22g) were divided into an experimental group and a blank control group. The experimental group was immunized twice by intramuscular injection of LNP containing 5μg of the above different mRNAs at 2-week intervals. Two weeks after each immunization, blood was collected from the orbital sinus of mice in the experimental group and the control group, and the serum was collected by centrifugation and stored at -20 degrees Celsius for later use. Two weeks after the second immunization, the spleens of mice in the experimental group and the control group were harvested for later use.

[0124] The serum from the experimental group mice was diluted at gradients of 10000×, 20000×, 40000×, 80000×, 160000×, 320000×, and 640000×, and added to ELISA plates pre-coated with standard gE protein, along with the serum from the negative control group. 100 μL of each sample was taken and three parallel replicates were performed. The plates were sealed with a sealing membrane and incubated at 4°C for 2 h. Remove the 96-well plate after primary antibody incubation, discard the primary antibody solution, and pat dry on absorbent paper until no residue remains. Add 100 μL of diluted secondary antibody (Goatpab to Ms IgG(HRP), 1:50000) to each well, seal the plate, and incubate at room temperature in the dark for 1 hour. Remove the plate after secondary antibody incubation, discard the secondary antibody solution, and pat dry on absorbent paper until no residue remains. Under room temperature and in the dark, remove the disposable sample container and pour TMB (3,3′,5,5′-Tetramethylbenzidine) solution into it. Add 100 μL of TMB to each well using a multi-channel pipette and incubate at room temperature in the dark for 20 minutes. Positive samples will turn blue at this time. Record the addition and color development times for each plate to ensure consistent incubation time. After the color development is complete, add 100 μL of Stop Solution to each well using a multi-channel pipette. At this time, the sample will turn yellow. Measure its absorbance at 450 nm. Read the plate within two minutes after adding the Stop Solution to each microplate.

[0125] Add mouse lymphocyte separation medium to a six-well plate, place a 70μm cell sieve inside, and place the spleen of an immunized mouse on the sieve. Grind the spleen using a syringe plunger until it becomes colorless. Transfer the lower cell suspension to a 15ml centrifuge tube, slowly add 1ml of RPMI-1640 medium to the cell suspension layer (without disrupting the interface), centrifuge at 800g for 30min at room temperature (Ultra 3, Dec 3), aspirate the lymphocyte layer, add 10mL of RPMI-1640, centrifuge at 350g for 10min at room temperature, discard the supernatant, and resuspend the cells in 2mL of RPMI-1640 complete medium (+10% inactivated serum + 1% penicillin antibiotics) to prepare spleen cell suspension. Add the spleen cells to a cell culture medium at 2×10⁻⁶ cells / mL. 6Each well contains 1 sample, and the culture medium is added to a final volume of 200 μL. The corresponding peptide library (2 μg / mL) or an equal volume of solvent is added for stimulation. After standing in a CO2 incubator at 37°C for 1 h, 0.4 μL of protein transport inhibitor is added to each well. The wells are then co-cultured at 37°C for 5 h and temporarily stored at 4°C overnight. Cells were then transferred from the wells to 2 mL centrifuge tubes and centrifuged at 350 g, 4 °C for 5 min. The supernatant was discarded, and 100 μL of Zombie staining solution was added to each well. After centrifugation, the supernatant was discarded again. Then, PBS containing 1% BSA and anti-mouse CD16 / CD32 (Biolegend) mAb, a mixture of 50 μL anti-mouse CD3 (Biolegend), anti-mouse CD4 (Biolegend), and anti-mouse CD8 (Biolegend), and a mixture of 150 μL LFixation / Permeabilization solution (Biolegend), and anti-mouse IFN-γ (Biolegend) and anti-mouse IL-2 (Biolegend) were added sequentially. The supernatant was discarded after each step. Finally, 200 μL of PBS containing 1% BSA was added, and the cells were analyzed by flow cytometry. Peptide-stimulated and unstimulated CD3+ cells were recorded. + CD4 + IFN-γ + CD3 + CD8 + IFN-γ + CD3 + CD4 + IL-2 + CD3 + CD8 + IL-2 + The cell proportions were determined and processed using CytExpert 2.4 software.

[0126] The results are as follows Figure 2 As shown in Tables 11 and 12.

[0127] Table 11. Antibody expression levels in experiments with different cap mRNAs

[0128]

[0129] Table 12. IL2 assays using mRNA with different caps + With IFN-g + CD4 + T cell kinase levels

[0130]

[0131] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention to achieve the same purpose. Therefore, all equivalent technical solutions also fall within the protection scope of this invention.

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 of 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 of any one of claims 1-4.

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

7. A method of in vitro co-transcriptional RNA capping, the method comprising performing template transcription in a transcription reaction system comprising the composition of 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℃.

Images