Capped composition as well as preparation method and in-vitro transcription reaction system thereof

Abstract

The invention provides a capping composition as well as a preparation method and an in-vitro transcription reaction system thereof. The composition is mainly composed of a salt solution formed by compounding a capping analogue and TRIS according to the molar ratio of 1: (3-7), as TRIS has good biocompatibility, when transcription is conducted on some special mRNA sequences, the biocompatibility of TRIS and mRNA is more excellent, the in-vitro transcription efficiency of mRNA can be improved, and the capping efficiency of mRNA can be obviously improved.

Description

A capping composition, preparation method thereof, and in vitro transcription reaction system Technical Field

[0001] The present invention relates to the fields of chemistry and bioengineering, and in particular to a capping composition, a preparation method thereof, and an in vitro transcription reaction system. Background Art

[0002] In vitro transcription of mRNA has become an important tool for introducing exogenous genes to express proteins and is widely used in the treatment and prevention of diseases. During in vitro transcription of mRNA, chemical capping of the mRNA is a critical step.

[0003] However, the system used for mRNA capping is relatively complex. For example, the recognition efficiency of capping enzymes for some specific mRNA sequences is poor, resulting in low capping efficiency. Some mRNA sequences also have a lot of secondary structure, which can cause significant steric hindrance during capping, ultimately leading to low capping efficiency.

[0004] Therefore, there is an urgent need in the art to develop a new class of capping analog combinations so that in vitro transcribed mRNA can achieve higher capping efficiency. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a capping composition. The application of TRIS in the capping composition can significantly improve the capping efficiency of mRNA.

[0006] A second object of the present invention is to provide a method for preparing a capping composition, which realizes the preparation of the capping composition, is simple to operate, and the prepared composition has stable performance.

[0007] A third object of the present invention is to provide an in vitro transcription reaction system containing the above-mentioned capping composition, wherein the mRNA synthesized by the transcription reaction has high capping efficiency and high yield; and compared with the control group of other types of capping compositions, the cell protein expression is significantly improved.

[0008] In a first aspect, the present application provides a capping composition, which adopts the following technical solution:

[0009] A capping composition mainly consists of a salt solution in which a capping analogue and TRIS are compounded in a molar ratio of 1:(3-7).

[0010] The inventors of the present application applied TRIS to a biological enzyme catalysis system. Specifically, a capping analog and TRIS were compounded in a molar ratio of 1:(3-7) to form a salt solution to obtain a capping composition. Because TRIS has relatively good biocompatibility, when transcribing certain special mRNA sequences, TRIS has even better biocompatibility with mRNA, which is beneficial for increasing the yield of mRNA transcription and significantly improving the mRNA capping efficiency.

[0011] Preferably, the structural formula of the capped analog is:

[0012] ;

[0013] The structural formula of TRIS is:

[0014] ;

[0015] Wherein, B1 and B2 are natural or modified bases, respectively;

[0016] R1 is H, OH, alkyl, O-alkyl or halogen;

[0017] R2 is H, OH, alkyl, O-alkyl or halogen;

[0018] R3 is hydrogen, hydroxy, or substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted N-dihydrocarbyl, substituted or unsubstituted O-aryl, substituted or unsubstituted S-aryl, substituted or unsubstituted NH-aryl, substituted or unsubstituted O-aralkyl, substituted or unsubstituted S-aralkyl, or substituted or unsubstituted NH-aralkyl;

[0019] R4 is H, OH, substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted N-dihydrocarbyl, substituted or unsubstituted O-aryl, substituted or unsubstituted S-aryl, substituted or unsubstituted NH-aryl, substituted or unsubstituted O-aralkyl, substituted or unsubstituted S-aralkyl, substituted or unsubstituted NH-aralkyl;

[0020] X1, X2 and X3 are O, CH2 or NH respectively;

[0021] Y1, Y2 and Y3 are O, S, Se or BH3 respectively.

[0022] Preferably, the capping efficiency of the capping composition reaches above 98%.

[0023] By adopting the above technical solution and the capping composition obtained by the formula and preparation method of the present application, the capping efficiency can reach more than 98%. Through experiments, it was found that the capping efficiency of the salt solution formed by compounding TRIS with the capping analogue was significantly improved compared with the sodium salt capping composition or the ammonium salt capping composition, which exceeded the inventor's expectations.

[0024] Preferably, when the capping composition is used to transcribe mRNA in vitro, the yield is increased by more than 45% compared with the sodium salt capping composition and the ammonium salt capping composition.

[0025] By adopting the above technical solution, when using a salt solution containing a capping analog and tris for in vitro transcription and synthesis of mRNA, the yield is increased by more than 45% compared to sodium salt capping compositions and ammonium salt capping compositions, which is a significant increase in yield. In some embodiments of the present application, the yield of synthesized mRNA using a salt solution containing tris reaches 72 μg, while the yield of synthesized mRNA using a sodium salt capping composition or an ammonium salt capping composition is less than 50 μg.

[0026] Preferably, after in vitro transcription, the capping composition improves the mRNA protein translation efficiency in cells by more than 1.25 times compared with the sodium salt capping composition and the ammonium salt capping composition.

[0027] Using the capping composition of the present application, after in vitro transcription, the efficiency of intracellular mRNA protein translation is increased by more than 1.25 times compared with the sodium salt capping composition or the ammonium salt capping composition, resulting in better intracellular protein expression and more excellent transcription and translation effects.

[0028] In a second aspect, the present application provides a method for preparing a capping composition, which adopts the following technical solution:

[0029] A method for preparing a capping composition comprises the following steps:

[0030] m7GDP imidazole salt was dissolved in a DMF solution containing MnCl2 and added to a DMF solution of 5'-phosphorylated dinucleotide. The reaction was stirred at room temperature and then concentrated to obtain the triethylamine salt form of the cap analogue.

[0031] converting the cap analog in triethylamine salt form to the cap analog in free acid form by passing it through a cation exchange resin;

[0032] The TRIS solution is added dropwise to the capped analog in free acid form, stirred evenly, and then concentrated to obtain a capped composition.

[0033] The triethylamine salt form of the cap analog is prepared using m7GDP imidazole salt and 5'-phosphorylated dinucleotide, and the triethylamine salt form of the cap analog is then converted into a free acid form of the cap analog through a cation exchange resin. TRIS solution is then added dropwise to the free acid form of the cap analog for reconstitution, and the mixture is stirred evenly. The amino groups in the TRIS solution are paired with the negatively charged phosphate groups in the nucleotide for positive and negative charge pairing, and the cap composition of the present application is prepared through a concentration process. The process is simple, and a pairing process with TRIS solution is added to the original preparation process of the cap analog. The resulting final product has high capping efficiency and excellent protein expression, transcription and translation effects in cells.

[0034] Preferably, the concentration of the TRIS salt solution is 0.3-0.7 mol / L.

[0035] By adopting the above technical solution, when the concentration of the TRIS salt solution is greater than 0.7 mol / L, a large amount of salt will remain in the final capping composition, which will inhibit the activity of the biological enzyme, reduce the in vitro transcription yield and the mRNA capping efficiency; when the concentration of the TRIS salt solution is less than 0.3 mol / L, the capping composition cannot be formed due to the low concentration of the TRIS salt solution.

[0036] Preferably, when the TRIS salt solution is added dropwise to the capped analog in free acid form, the solution is added dropwise until the pH of the TRIS salt solution reaches 3-7.

[0037] By adopting the above technical solution, when the TRIS solution is added dropwise, the titration can be stopped when the pH of the solution containing the capping composition is between 3 and 7. At this time, a capping composition with stable performance can be prepared; if the pH is greater than 7 or the pH is less than 3, the structural sites on the phosphate group are easily decomposed, which can easily cause the performance of the capping composition to be unstable and be detrimental to the activity of the capping enzyme during mRNA transcription.

[0038] In a third aspect, the present application provides an in vitro transcription reaction system, which adopts the following technical solution:

[0039] An in vitro transcription reaction system contains RNA polymerase, nucleoside triphosphates and the above-mentioned capping composition.

[0040] By adopting the above technical solution, RNA polymerase, nucleoside triphosphates and capping composition constitute the in vitro transcription reaction system of the present application. After transcription by the in vitro transcription system, the mRNA yield and capping efficiency are significantly improved.

[0041] Preferably, the nucleoside triphosphate may be a natural base nucleoside triphosphate, a modified nucleoside triphosphate or a non-natural nucleoside triphosphate.

[0042] Preferably, the RNA polymerase is an RNA polymerase derived from bacteriophage.

[0043] Further preferably, the RNA polymerase is selected from T7, SP6 or T3.

[0044] More preferably, the RNA polymerase is selected from T7.

[0045] In summary, this application has at least the following technical effects:

[0046] 1. This application is to compound a capping analog with TRIS to form a salt solution, that is, to obtain a capping composition. When transcribing certain special mRNA sequences, TRIS and mRNA have better biocompatibility, which is beneficial to increasing the yield of mRNA transcription and significantly improving the mRNA capping efficiency;

[0047] 2. This application limits the concentration range of the added TRIS solution. If the concentration exceeds the range by a large amount, a large amount of salt will remain in the final capping composition, which will inhibit the activity of the biological enzyme and reduce the capping efficiency. If the concentration exceeds the range by a small amount, the capping composition cannot be formed due to the low concentration of the TRIS salt solution.

[0048] 3. The inventors have discovered through experiments that the use of TRIS in combination with a capping analog significantly improves the capping efficiency during in vitro transcription and synthesis of mRNA, compared to sodium salt capping compositions or ammonium salt capping compositions, and increases the mRNA yield by more than 45%.

[0049] 4. The inventors discovered through experiments that the use of TRIS in combination with a capping analogue increased intracellular protein expression by more than 1.25 times compared to a sodium salt capping composition or an ammonium salt capping composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG1 is a schematic diagram of a reaction structure containing 2 to 3 tris groups in Example 1.

[0051] FIG2 is a schematic diagram of the reaction structure of Example 2 containing 4 to 5 tris.

[0052] FIG3 is a schematic diagram of the reaction structure of Example 3 containing 6 to 7 tris.

[0053] FIG4 is a schematic diagram showing the reaction structure of Comparative Example 1 containing 2 to 3 sodium ions.

[0054] FIG5 is a schematic diagram showing the reaction structure of Comparative Example 2 containing 4 to 5 sodium ions.

[0055] FIG6 is a schematic diagram showing the reaction structure of Comparative Example 3 containing 6 to 7 sodium ions.

[0056] FIG7 is a schematic diagram showing the reaction structure of Comparative Example 4 containing 4 to 5 ammonium cations.

[0057] FIG8 is a schematic diagram illustrating the intracellular mRNA protein translation effects of Example 1, Comparative Example 1, and Comparative Example 4. Implementation Method

[0058] The present invention is further described in detail below with reference to Figures 1 to 7 and Examples. All raw materials involved in the Examples and Comparative Examples of the present invention are commercially available. Example 1:

[0059] A capping composition, in conjunction with Figure 1, is prepared by the following method:

[0060] I: Compound m7GDP imidazole salt (2 mmol) was dissolved in a DMF solution containing MnCl2 (2 mmol) and added to a DMF solution of 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt). The reaction was stirred at room temperature. After 24 hours, the reaction was terminated with 10 mL of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column (3×50 cm). The product was eluted using a linear gradient of 0-1.0 M TEAB eluent. The eluted product with an HPLC purity of >97% was collected and concentrated to obtain a triethylamine salt form (c) of the cap analog at a concentration of 120 mM.

[0061] II: converting the triethylamine salt form of the cap analog to the free acid form at the same concentration through a cation exchange resin;

[0062] III: Add 0.2 M TRIS base to the product solution until the pH reaches 3-3.6, then concentrate and adjust the volume to obtain the final product, which now has a morphology of 2-3 tris counter ions. Example 2:

[0063] A capping composition, in conjunction with Figure 2, is prepared by the following method:

[0064] I: Compound m7GDP imidazole salt (2 mmol) was dissolved in a DMF solution containing MnCl2 (2 mmol) and added to a DMF solution of 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt). The reaction was stirred at room temperature. After 24 hours, the reaction was terminated with 10 mL of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column (3×50 cm). The product was eluted using a linear gradient of 0-1.0 M TEAB eluent. The eluted product with an HPLC purity of >97% was collected and concentrated to obtain a triethylamine salt form of the cap analog at a concentration of 120 mM;

[0065] II: converting the triethylamine salt form of the cap analogue into the free acid form at the same concentration through a cation exchange resin;

[0066] III: Add 0.2M TRIS base dropwise to the product solution until the pH reaches 5-5.8, then concentrate and adjust the volume to obtain the final product, which now has a system of 4-5 tris counter ions. Example 3:

[0067] A capping composition, in conjunction with FIG3 , is prepared by the following method:

[0068] I: Compound m7GDP imidazole salt (2 mmol) was dissolved in a DMF solution containing MnCl2 (2 mmol) and added to a DMF solution of 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt). The reaction was stirred at room temperature. After 24 hours, the reaction was terminated with 10 mL of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column (3×50 cm). The product was eluted using a linear gradient of 0-1.0 M TEAB eluent. The eluted product with an HPLC purity of >97% was collected and concentrated to obtain a triethylamine salt form of the cap analog at a concentration of 120 mM;

[0069] II: converting the triethylamine salt form (c) of the cap analog to the free acid form at the same concentration through a cation exchange resin;

[0070] III: 0.2M TRIS base was added dropwise to the product solution until the pH was 6-7.2, and then the solution was concentrated and fixed to volume to obtain the final product, which now had a morphology of 6-7 tris counter ions.

[0071] Comparative Example 1:

[0072] A sodium salt capping composition, in conjunction with Figure 4, is prepared by the following method:

[0073] I: Compound m7GDP imidazole salt (2 mmol) was dissolved in a DMF solution containing MnCl2 (2 mmol) and added to a DMF solution of 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt). The reaction was stirred at room temperature; after 36 hours, the reaction was terminated with 10 mL of 0.25 M EDTA solution; the mixture was loaded onto a DEAE Sephadex column (3 × 50 cm); the product was eluted using a linear gradient of 0-1.0 M TEAB eluent. The eluted product with an HPLC purity of >97% was collected, concentrated, and desalted to obtain the triethylamine salt form of the cap analog;

[0074] II: converting the triethylamine salt form of the cap analog to the free acid form at the same concentration through a cation exchange resin;

[0075] III: 0.1 M NaOH solution was added dropwise to the product solution to adjust the pH to 3-3.6, and then concentrated and fixed to volume to obtain a 100 mM final product containing 2-3 sodium ion counterions.

[0076] Comparative Example 2:

[0077] A sodium salt capping composition, in conjunction with Figure 5, is prepared by the following method:

[0078] I: Compound m7GDP imidazole salt (2 mmol) was dissolved in a DMF solution containing MnCl2 (2 mmol) and added to a DMF solution of 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt). The reaction was stirred at room temperature; after 36 hours, the reaction was terminated with 10 mL of 0.25 M EDTA solution; the mixture was loaded onto a DEAE Sephadex column (3 × 50 cm); the product was eluted using a linear gradient of 0-1.0 M TEAB eluent. The eluted product with an HPLC purity of >97% was collected, concentrated, and desalted to obtain the triethylamine salt form of the cap analog;

[0079] II: converting the triethylamine salt form of the cap analog to the free acid form at the same concentration through a cation exchange resin;

[0080] III: 0.1 M NaOH solution was added dropwise to the product solution to adjust the pH to 5-5.8, and then concentrated and fixed to volume to obtain a 100 mM final product containing 4-5 sodium ion counterions.

[0081] Comparative Example 3:

[0082] A sodium salt capping composition, in conjunction with Figure 6, is prepared by the following method:

[0083] I: Compound m7GDP imidazole salt (2 mmol) was dissolved in a DMF solution containing MnCl2 (2 mmol) and added to a DMF solution of 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt). The reaction was stirred at room temperature; after 36 hours, the reaction was terminated with 10 mL of 0.25 M EDTA solution; the mixture was loaded onto a DEAE Sephadex column (3 × 50 cm); the product was eluted using a linear gradient of 0-1.0 M TEAB eluent. The eluted product with an HPLC purity of >97% was collected, concentrated, and desalted to obtain the triethylamine salt form of the cap analog;

[0084] II: converting the triethylamine salt form of the cap analog to the free acid form at the same concentration through a cation exchange resin;

[0085] III: 0.1 M NaOH solution was added dropwise to the product solution to adjust the pH to 6-7.2, and then concentrated and fixed to volume to obtain a 100 mM final product containing 6-7 sodium ion counterions.

[0086] Comparative Example 4:

[0087] An ammonium salt capping composition, in conjunction with FIG7 , is prepared by the following method:

[0088] I: Compound m7GDP imidazole salt (2 mmol) was dissolved in a DMF solution containing MnCl2 (2 mmol) and added to a DMF solution of 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt). The reaction was stirred at room temperature; after 36 hours, the reaction was terminated with 10 mL of 0.25 M EDTA solution; the mixture was loaded onto a DEAE Sephadex column (3 × 50 cm); the product was eluted using a linear gradient of 0-1.0 M TEAB eluent. The eluted product with an HPLC purity of >97% was collected, concentrated, and desalted to obtain the triethylamine salt form of the cap analog;

[0089] II: converting the triethylamine salt form of the cap analog to the free acid form at the same concentration through a cation exchange resin;

[0090] III: 0.5 M ammonia solution was added dropwise to the product solution to adjust the pH to 5.8-6.2, and then concentrated and fixed to volume to obtain a 100 mM final product containing 4-5 ammonium cation counterions.

[0091] Comparative Example 5:

[0092] A capping composition is prepared by the following method:

[0093] I: Compound m7GDP imidazole salt (2 mmol) was dissolved in a DMF solution containing MnCl2 (2 mmol) and added to a DMF solution of 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt). The reaction was stirred at room temperature. After 24 hours, the reaction was terminated with 10 mL of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column (3×50 cm). The product was eluted using a linear gradient of 0-1.0 M TEAB eluent. The eluted product with an HPLC purity of >97% was collected and concentrated to obtain a triethylamine salt form of the cap analog at a concentration of 120 mM.

[0094] II: converting the triethylamine salt form of the cap analog to the free acid form at the same concentration through a cation exchange resin;

[0095] III: Add 0.2 M TRIS base to the product solution until the pH reaches 2.3-2.8, then concentrate and adjust the volume to obtain the final product, which now has a 1-2 tris counterion system.

[0096] Comparative Example 6:

[0097] A capping composition is prepared by the following method:

[0098] I: Compound m7GDP imidazole salt (2 mmol) was dissolved in a DMF solution containing MnCl2 (2 mmol) and added to a DMF solution of 5'-phosphorylated dinucleotide (1 mmol; triethylammonium salt). The reaction was stirred at room temperature. After 24 hours, the reaction was terminated with 10 mL of 0.25 M EDTA solution. The mixture was loaded onto a DEAE Sephadex column (3×50 cm). The product was eluted using a linear gradient of 0-1.0 M TEAB eluent. The eluted product with an HPLC purity of >97% was collected and concentrated to obtain a triethylamine salt form of the cap analog at a concentration of 120 mM.

[0099] II: converting the triethylamine salt form of the cap analog to the free acid form at the same concentration through a cation exchange resin;

[0100] III: Add 0.2 M TRIS base to the product solution until the pH reaches 7.5-8.2, then concentrate and adjust the volume to obtain the final product, which now has a system of 7-8 tris counter ions.

[0101] The biological indicators such as IVT yield, capping efficiency, and intracellular target protein expression of three cap analogs of sodium salt capping composition, ammonium salt capping composition, and capping composition were compared.

[0102] In vitro synthesis of mRNA using capping compositions

[0103] (1) Preparation of DNA template

[0104] The usual template is a linearized plasmid, a PCR amplification product or a synthetic DNA fragment. For the preparation of a linearized plasmid template, the target fragment is usually inserted into the multiple cloning site of the plasmid using methods such as enzyme cutting or recombination. The plasmid generally has a start site for the corresponding RNA polymerase, such as T7, SP6 or T3, and then amplified in a "biofactory" such as Escherichia coli. The extracted and purified modified plasmid is cut and purified using a restriction endonuclease and can be used as a DNA template for in vitro transcription of mRNA. For the preparation of PCR amplification products, the target DNA fragment is usually transcribed using a DNA polymerase, specific primers and related buffers. The amplified product usually contains a start site for the corresponding RNA polymerase, such as T7, SP6 or T3. In this embodiment, T7 RNA polymerase is selected. After purification, the amplified product can be used as a DNA template for in vitro transcription of mRNA.

[0105] (2) In vitro transcription system

[0106] Table 1 Configuration of mRNA in vitro transcription system

[0107] The capping compositions are: sodium salt capping composition, ammonium salt capping composition, and tris salt.

[0108] During the experiment, sterile, enzyme-free water was first added to the system, followed by 10X buffer, ATP, GTP, CTP, N1-Me-pUTP, and capping composition. Mix thoroughly and then gently centrifuge. Nuclease inhibitors, inorganic pyrophosphatase, T7 RNA polymerase, and DNA template were then added. Mix thoroughly, centrifuge, and incubate at 37°C. After 2 hours, 1 unit of DNase I was added, and incubation at 37°C for another 30 minutes to remove the DNA template. RNA was then purified.

[0109] (3) mRNA yield

[0110] The purified mRNA was dissolved in sterile enzyme-free water and then quantitatively detected using a UV spectrophotometer NanodropOne. The results are shown in Table 2.

[0111] Table 2 Yields of in vitro transcribed mRNA from different starting capping compositions

[0112] Combining the test results of Examples 1 to 3, Comparative Examples 1 to 4, and Table 2, it can be seen that when the capping composition of the present application is used for in vitro transcription and synthesis of mRNA, the mRNA yield is significantly increased, reaching 72 to 74 μg, while the sodium salt capping composition or the ammonium salt capping composition is used. The mRNA yield is significantly reduced, and the yield is reduced to 47 to 49 μg. The experimental results show that the composition of the present application is used for in vitro transcription, and the yield of synthesized mRNA is increased by 46.9% to 57.4%, which greatly exceeds the experimental expectations.

[0113] Combining the test results of Examples 1-3, Comparative Examples 5-6, and Table 2, it can be seen that when the pH of the capping composition exceeds the range of 3-7, that is, when the molar ratio of the cap analog to TRIS exceeds the range of 1:(3-7), the mRNA yield decreases significantly. This is because the structural sites on the phosphoside are easily decomposed, resulting in the unstable performance of the capping composition, which is not conducive to the activity of the biological enzyme during mRNA transcription.

[0114] (4) Comparison of capping efficiency

[0115] Liquid chromatography-mass spectrometry (LC-MS) is used to measure the IVT capping efficiency of mRNA with different initiation cap analogs. First, a labeled DNA probe is designed that matches the start base of the transcript, typically with a biotin label. After washing, the streptavidin-labeled magnetic beads are incubated with the synthesized DNA probe, mRNA, and 10× RNase H reaction buffer at room temperature for 30 minutes, gently mixing. Subsequently, 20 μL of RNase H (5 U / μL) is added and incubated at 37°C for 3 hours, mixing every half hour. After incubation, the beads are washed and 100 μL of 75% methanol heated to 80°C is added. The mixture is heated to 80°C on a hot plate for 3 minutes, then placed on a magnetic rack. The supernatant is aspirated and dried in an evaporating centrifuge at room temperature for 45 minutes to a volume of 10 μL. The sample was then resuspended in 50 μl of 100 μM EDTA / 1% MeOH and used for LC-MS analysis to determine RNA capping during the transcription reaction. Because capped and uncapped bases differ significantly in molecular weight, this molecular mass difference can be used to determine the capping efficiency of mRNA transcripts initiated with different cap analogs. Detailed results from the same batch are shown in Table 3 and the mass spectra.

[0116] Table 3 Capping efficiency of in vitro transcribed mRNA with different starting capping compositions

[0117] Combined with the test results of Examples 1 to 3, Comparative Examples 1 to 4, and Table 3, it can be seen that when the capping composition of the present application is used for in vitro transcription and synthesis of mRNA, the capping efficiency of mRNA can reach 99.7%, while the sodium salt capping composition or the ammonium salt capping composition is used. The capping efficiency is significantly reduced to below 95%. The experimental results show that the capping efficiency of mRNA is significantly improved when the composition of the present application is used for in vitro transcription, far exceeding the expectations of the inventors.

[0118] Combining the test results of Examples 1 to 3, Comparative Examples 5 to 6, and Table 3, it can be seen that when the pH of the capping composition exceeds the range of 3 to 7, that is, when the molar ratio of capping agent to TRIS exceeds the range of 1:(3 to 7), the mRNA capping efficiency decreases significantly. This is because the structural sites on the phosphoside are easily decomposed, resulting in the unstable performance of the capping composition, which is not conducive to the activity of the capping enzyme during mRNA transcription, thereby affecting the improvement of the mRNA capping efficiency.

[0119] (5) Cell protein expression

[0120] In this example, the eGFP coding sequence was used as a DNA template, and the capping compositions of Comparative Example 1, Comparative Example 4, and Example 1 were selected as starting materials for in vitro transcription. The specific capping steps were: m 7 GpppA 2’O m e The different mRNA products were then transfected into 293T cells. Specifically, 293T cells were transfected with 0.5-1×10 5 Cells were plated in 24-well plates. Cells within 50 passages are recommended for transfection experiments. The cells should be passaged again 24 hours before transfection. The addition of antibiotics to the culture medium has no effect on transfection efficiency. A cell density of 60-80% is generally optimal for transfection. 2 μg of mRNA was transfected per well using Lipofectamine Messenger MAX Transfection Reagent (Invitrogen) and following the instructions. After transfection, the cells were placed in a 37°C, CO2 incubator. After 4-6 hours of transfection, the culture medium was replaced with fresh complete medium. After 24 hours of incubation at 37°C in a CO2 incubator, GFP fluorescence intensity was observed under a fluorescence microscope.

[0121] Combining the intracellular mRNA protein translation effects shown in Example 1, Comparative Example 1, Comparative Example 4 and Figure 8, it can be seen that the mRNA protein translation effect obtained by in vitro transcription using the capping composition of the present application is more than 1.5 times that of the sodium salt capping composition and more than 1.25 times that of the ammonium salt capping composition. It can be seen that when the TRIS-compounded capping composition is used for in vitro transcription, the mRNA expression effect and translation effect in the cell are better.

Claims

1. A capping composition, characterized in that: The composition mainly consists of a salt solution in which the capping analogue and TRIS are compounded in a molar ratio of 1:(3-7).

2. The capping composition according to claim 1, wherein: The structural formula of the capped analog is: ； The structural formula of TRIS is: ； Wherein, B1 and B2 are natural or modified bases, respectively; R1 is H, OH, alkyl, O-alkyl or halogen; R2 is H, OH, alkyl, O-alkyl or halogen; R3 is hydrogen, hydroxy, or substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted N-dihydrocarbyl, substituted or unsubstituted O-aryl, substituted or unsubstituted S-aryl, substituted or unsubstituted NH-aryl, substituted or unsubstituted O-aralkyl, substituted or unsubstituted S-aralkyl, or substituted or unsubstituted NH-aralkyl; R4 is H, OH, substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted N-dihydrocarbyl, substituted or unsubstituted O-aryl, substituted or unsubstituted S-aryl, substituted or unsubstituted NH-aryl, substituted or unsubstituted O-aralkyl, substituted or unsubstituted S-aralkyl, substituted or unsubstituted NH-aralkyl; X1, X2 and X3 are O, CH2 or NH respectively; Y1, Y2 and Y3 are O, S, Se or BH3 respectively.

3. A capping composition according to claim 2, characterized in that: The capping efficiency of the capping composition reaches more than 98%.

4. The capping composition according to claim 2, wherein: When the capping composition is used to transcribe mRNA in vitro, the yield is increased by more than 45% compared with the sodium salt capping composition and the ammonium salt capping composition.

5. The capping composition according to claim 2, wherein: After in vitro transcription, the capping composition improves the mRNA intracellular protein translation efficiency by more than 1.25 times compared with the sodium salt capping composition and the ammonium salt capping composition.

6. The method for preparing a capping composition according to any one of claims 1 to 5, wherein: The steps include: m7GDP imidazole salt was dissolved in a DMF solution containing MnCl2 and added to a DMF solution of 5'-phosphorylated dinucleotide. The reaction was stirred at room temperature and then concentrated to obtain the triethylamine salt form of the cap analogue. converting the cap analog in triethylamine salt form to the cap analog in free acid form by passing it through a cation exchange resin; The TRIS solution is added dropwise to the capped analog in free acid form, stirred evenly, and then concentrated to obtain a capped composition.

7. The method for preparing a capping composition according to claim 6, wherein: The concentration of the TRIS solution is 0.3-0.7 mol / L.

8. The method for preparing a capping composition according to claim 6, wherein: When the TRIS solution is added dropwise to the cap analog in the free acid form, the pH of the complex salt solution is added dropwise to 3-7.

9. An in vitro transcription reaction system, characterized in that: The in vitro transcription reaction system contains RNA polymerase, nucleoside triphosphates and the capping composition according to any one of claims 1 to 5.

10. An in vitro transcription reaction system according to claim 9, characterized in that: The nucleoside triphosphate is selected from natural base nucleoside triphosphate, modified nucleoside triphosphate or non-natural nucleoside triphosphate; the RNA polymerase is selected from T7, SP6 or T3.

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