Base-modified mRNA (messenger Ribonucleic Acid) and application thereof in cell-free protein synthesis system

Abstract

The invention discloses base-modified mRNA (messenger Ribonucleic Acid) and application thereof in a cell-free protein synthesis system, and belongs to the technical field of nucleic acid modification. Natural nucleoside triphosphate is completely replaced by different types of basic group modified nucleoside triphosphate, mRNA with different chemical modifications is obtained through an in-vitro transcription method and purification, and the mRNA is put into an in-vitro cell-free protein synthesis system for reaction. A reaction element recombination system-PURE system based on prokaryote Escherichia coli purified protein and an eukaryote CFPS system taking rabbit reticulated red lysate as a material are mainly selected to detect the corresponding translation efficiency of mRNA modified by different bases in two cell-free protein synthesis systems respectively. Different regulatory activities may be shown in different cell-free protein synthesis systems when different modified nucleotides are doped into mRNA, base modified triphosphoric acid capable of improving the translation efficiency of an in-vitro protein synthesis system is screened out, and the stability of mRNA in the cell-free protein synthesis system and the corresponding protein expression level are improved.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid modification technology, specifically relating to a base-modified mRNA and its application in a cell-free protein synthesis system. Background Technology

[0002] Cell-free protein synthesis (CFPS) is a method for rapidly synthesizing target proteins in vitro, also known as in vitro protein transcription and translation technology. CFPS systems use cell extracts to simulate the intracellular environment, freeing researchers from the constraints of intact living cells. This highly open synthetic system allows for direct control of transcription and translation processes in vitro, providing significant convenience and flexibility for protein synthesis research. With the rapid development of CFPS, the types of CFPS systems have become increasingly diverse. Currently, they are mainly divided into two categories: one is extract systems, primarily including E. coli, yeast, and rabbit reticulocyte extract systems. These systems utilize cell extracts from different sources, preserving some key intracellular transcription and translation machinery, enabling protein synthesis in vitro. The other category is cell-free transcription and translation systems that synthesize proteins using recombinant elements, namely the PURE system. The PURE system is constructed using purified recombinases and factors, offering advantages such as well-defined components and precise controllability, providing an ideal model for in-depth research on protein synthesis mechanisms.

[0003] While cell-free protein synthesis systems offer significant advantages such as speed and efficiency, their open nature also presents a series of challenges. Because the components in the system are directly exposed to the external environment and their concentrations are artificially controlled, protein synthesis is easily affected and disrupted by environmental or human interference. Among these challenges, the stability of the genetic template (including DNA and mRNA templates) is a key factor limiting the efficiency of protein synthesis. The presence of nucleases in cell-free protein synthesis systems is a significant concern. Templates added to the cell-free reaction system, whether DNA or mRNA, face the risk of degradation by nucleases, ultimately affecting the overall protein synthesis efficiency of the expression system. This is especially true for mRNA encoding the target protein, which is one of the most vulnerable components in the entire reaction system. mRNA in the system is highly susceptible to degradation by nucleases, preventing it from repeatedly participating in the translation process. The quantity and stability of mRNA have a crucial impact on protein synthesis efficiency and yield. Studies have shown that in eukaryotic cell-free protein synthesis systems, such as the rabbit reticulin cell-free protein expression system, linear mRNA is more fragile and less stable than DNA templates, often resulting in lower yields in eukaryotic cell-free protein synthesis systems.

[0004] Natural mRNA molecules have several inherent limitations. They are easily degraded by nucleases in the in vivo environment, have a short half-life, and can stimulate the activation of cellular innate immunity, triggering an immune response. This further restricts their application in the biomedical field. In eukaryotes, numerous studies have confirmed that chemical modification methods can improve mRNA performance. For example, incorporating modified nucleosides, such as pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ), during in vitro transcription (IVT) of mRNA can significantly reduce the immunogenicity of mRNA, enhance its stability, and improve protein expression. Taking the N1-methylpseudouridine-modified COVID-19 mRNA vaccine as an example, replacing the conventional UTP with N1-methylpseudouridine reduces the immunogenicity of the generated mRNA, improves protein expression, and provides favorable conditions for efficient mRNA delivery. In prokaryotes, related studies have also explored the chemical modification of mRNA. For example, messenger RNA (mRNA) was prepared by introducing phosphate thioester modification into mRNA, resulting in all 16 possible combinations of phosphate thioester modifications to the A, G, C, and U phosphate sites (PS-mRNA). The translation response was then evaluated using an E. coli cell-free translation system. These results demonstrate the significant potential of chemical modification in improving mRNA performance.

[0005] Currently, most modified nucleic acid types are used in in vivo cellular environments for research, while fewer modified nucleic acid types are applied to cell-free protein synthesis systems. Although some studies have demonstrated the effectiveness of chemical modification in improving mRNA stability and translation efficiency, a systematic screening and optimization method is still lacking in cell-free protein synthesis systems to identify modified triphosphates that can significantly improve the translation efficiency of in vitro protein synthesis systems. To address this issue, it is necessary to screen for corresponding modified triphosphates that enhance the translation efficiency of in vitro protein synthesis systems and introduce them into mRNA, thereby improving the stability of in vitro mRNA and enhancing its translational activity. This would supplement or enrich existing mRNA chemical modification strategies for cell-free in vitro translation systems, thus facilitating the application of mRNA chemical modification and cell-free protein synthesis. This will not only help improve the efficiency and yield of cell-free protein synthesis but also provide strong technical support and innovative ideas for the widespread application of mRNA chemical modification and cell-free protein synthesis technologies in biomedicine, bioengineering, and other fields. Summary of the Invention

[0006] The current state of affairs in cell-free protein synthesis systems suffers from poor genetic template stability, particularly the susceptibility of mRNA to nuclease degradation, which negatively impacts protein synthesis efficiency. While much research on modified nucleic acids has focused on in vivo delivery systems (such as mRNA vaccines or nucleic acid drugs), demonstrating that chemical modifications (e.g., 2'-O-methylation, pseudouridine) can enhance nucleic acid stability, a systematic screening and optimization method is lacking for cell-free protein synthesis systems. This invention aims to provide a base-modified mRNA and its application in cell-free protein synthesis systems. By chemically synthesizing non-natural modified nucleotides, the types and range of non-natural nucleosides can be enriched. Furthermore, by using a series of chemically modified nucleotides as raw materials and obtaining mRNAs with different chemical modifications through in vitro transcription, and then incorporating them into an in vitro cell-free protein synthesis system, it is possible to screen for corresponding modified triphosphates that improve the translation efficiency of the cell-free protein synthesis system to a certain extent. This enhances the translation efficiency and stability of the target mRNA, thereby increasing its corresponding protein expression level, providing a new option for mRNA chemical modification and improving the protein synthesis capacity of cell-free protein synthesis systems.

[0007] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a base-modified mRNA, which is obtained by in vitro transcription and purification of natural nucleotides completely replaced by chemically modified nucleotides; The chemically modified nucleotides are obtained by chemical synthesis or enzymatic synthesis of the bases and / or sugars of the modified nucleotides; The chemically modified nucleotides include chemically modified compounds of adenine, guanine, cytosine, and uracil, their salts, or isomers thereof.

[0008] The chemically modified nucleotide is modified by introducing any one or a combination of hydrogen, C2-C5 fatty acyl, aromatic acyl, haloacyl, C1-C5 alkyl, methoxy, halogen and nitrile groups into the base of the nucleotide, and by introducing hydroxyl, hydrogen, halogen, methoxy, ethoxy or methoxymethyl ether substituents into the sugar position of the nucleotide.

[0009] The chemically modified nucleotide is selected from one or more of N1-methylpseuuridine, thiophosphate modification, N6-methyladenine, 5-methylcytosine, N4-ethylcytosine, 2-thioguanine, and 5-methyluracil.

[0010] The mRNA contains at least one or more of the following structural elements: a promoter; or a 5'UTR containing at least one Kozak sequence, or a 3'UTR, or a coding sequence consisting of linked nucleosides, or a poly-A tail.

[0011] The purification process is performed by selective adsorption or high-performance liquid chromatography.

[0012] This invention provides the application of the aforementioned base-modified mRNA in improving the translation efficiency of cell-free protein synthesis systems.

[0013] This invention provides a method for improving the translation efficiency of a cell-free protein synthesis system. The method involves using a base-modified mRNA to participate in the cell-free protein synthesis process, and employing at least one of Western blot, enzyme-linked immunosorbent assay (ELISA), or radioisotope labeling to detect translation efficiency. The method then screens and optimizes mRNA stability and protein expression levels, establishing an optimization strategy specifically for the cell-free protein synthesis system.

[0014] Preferably, the expression efficiency of mRNA in a cell-free protein synthesis system is detected.

[0015] The present invention provides a cell-free protein synthesis system containing the aforementioned base-modified mRNA.

[0016] The system is either the PURE system of prokaryotic Escherichia coli or a eukaryotic system using rabbit reticulin lysate as material.

[0017] The aforementioned base-modified mRNA, or the aforementioned method for improving the translation efficiency of a cell-free protein synthesis system, or the aforementioned application of a cell-free protein synthesis system in vaccine antigen preparation, artificial biological system construction, or synthesis of therapeutic proteins.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The base-modified mRNA provided by this invention is obtained by in vitro transcription and purification of chemically modified nucleotides. The chemically modified nucleotides endow the mRNA with significantly enhanced stability compared to natural mRNA, laying the foundation for improving the translation efficiency of cell-free protein synthesis systems. Through in vitro transcription and purification, it helps to play a better role in cell-free protein synthesis systems.

[0019] Preferably, by introducing multiple groups at the base positions of natural nucleotides and different substituents at the glycosyl positions, the chemical properties and spatial structure of nucleotides can be altered, enhancing the resistance of mRNA to nucleases and reducing the likelihood of degradation in cell-free protein synthesis systems. This improves the stability of mRNA and provides a stable template for efficient protein synthesis. Cell-free protein synthesis systems are highly sensitive to reaction conditions (such as pH, temperature, and ionic strength), and the modified nucleotides can optimize thermal stability and anti-degradation capabilities to ensure efficient participation in protein synthesis in the in vitro environment.

[0020] The application provided by this invention, by using base-modified mRNA, can solve the problem of poor genetic template stability, reduce mRNA degradation by nucleases, and thus improve translation efficiency; it provides an effective means for optimizing cell-free protein synthesis systems, and helps to improve the yield and quality of protein synthesis in practical applications.

[0021] Furthermore, in traditional cell-free protein synthesis systems, exogenous mRNA is easily degraded by endogenous nucleases. By introducing chemical modifications such as 2'-O-acetyl groups and thiophosphates, the degradation rate of mRNA can be significantly reduced. Cell-free protein synthesis systems do not require cell culture and can directly and rapidly screen for optimal translation templates by replacing and selecting modified nucleotides. Cell-free protein synthesis systems can express toxic proteins (such as antimicrobial peptides and membrane proteins) that are difficult to synthesize in traditional cellular systems. Chemical modifications enhance ribosome binding efficiency by adjusting base pairing and stacking. Chemically modified mRNA can bind to lipid nanoparticles (LNPs) or polymer micelles for targeted delivery. Chemical modifications reduce PRR binding by altering RNA structure. Chemically modified mRNA, with its Cap1 cap structure (such as ARCA) and long polyA tail (>100 nt), resists degradation by uncapping enzymes and nucleases. Cell-free protein synthesis systems can tolerate a high proportion of modified nucleotides (such as full m1Ψ modification), while traditional cellular systems are prone to expression failure due to metabolic stress.

[0022] The method for improving the translation efficiency of cell-free protein synthesis systems provided by this invention uses base-modified mRNA to participate in the synthesis process and employs multiple detection methods to detect translation efficiency. By screening and optimizing mRNA stability and protein expression levels, an optimization strategy for cell-free protein synthesis systems is established. This method is systematic and targeted, and can comprehensively evaluate the impact of different modified mRNAs on system performance, thereby finding the most suitable modification mode for cell-free protein synthesis systems, effectively improving translation efficiency, and opening up new pathways for mRNA modification and enhancing the protein synthesis capabilities of cell-free protein synthesis systems.

[0023] The cell-free protein synthesis system provided by this invention introduces base-modified mRNA with good stability and translation efficiency into the cell-free protein synthesis system, which can improve the properties of the genetic template in the system, enhance the overall system's resistance to nucleases, reduce mRNA degradation, and thus enhance the system's protein synthesis capacity, providing a more reliable system platform for efficient protein synthesis.

[0024] The application provided by this invention, based on base-modified mRNA, can improve the translation efficiency and protein expression level of cell-free protein synthesis systems. Therefore, it can provide a more efficient and reliable protein synthesis solution for vaccine antigen preparation, artificial biological system construction, and synthesis of therapeutic proteins, and has broad application prospects and important social significance. Attached Figure Description

[0025] Figure 1 This is a flowchart of the method for improving the translation efficiency of a cell-free protein synthesis system based on chemically modified nucleoside triphosphates according to the present invention; Figure 2 This is a schematic diagram showing the results of in vitro transcription of prokaryotic mRNA with different chemically modified nucleoside triphosphates according to the present invention; Figure 3 This is a graph showing the expression efficiency of the mRNA transcribed in this invention in the PURE system for BFP protein. Figure 4 This is a schematic diagram showing the results of in vitro transcription of prokaryotic mRNA using different chemically modified nucleoside triphosphates according to the present invention; Figure 5 This is a graph showing the expression efficiency of the mRNA transcribed in this invention in the PURE system for sfGFP protein. Figure 6 This is a schematic diagram showing the results of in vitro transcription of eukaryotic mRNA using chemically modified A-series nucleoside triphosphates according to the present invention; Figure 7 This is a graph showing the expression efficiency of the mRNA transcribed in this invention in the rabbit reticulum lysis buffer system for luciferase protein. Figure 8 This is a schematic diagram showing the results of in vitro transcription of eukaryotic mRNA using chemically modified C-series nucleoside triphosphates according to the present invention. Figure 9 The expression efficiency of the mRNA transcribed in this invention in the rabbit reticulum lysis buffer system for luciferase protein; Figure 10 This is a schematic diagram showing the results of in vitro transcription of eukaryotic mRNA using chemically modified U-series nucleoside triphosphates according to the present invention. Figure 11 The expression rate of luciferase protein in the rabbit reticulin lysis buffer system is shown in the mRNA transcribed in this invention. Detailed Implementation

[0026] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0027] The present invention will now be described in further detail with reference to the accompanying drawings: Example 1 See appendix Figure 1 This embodiment provides a method for improving the translation efficiency of a cell-free protein synthesis system based on base-modified mRNA. 1. Design and synthesize the corresponding eukaryotic DNA templates. The design includes a DNA template containing a promoter sequence, a 5' UTR, and a 3' UTR, where the 5' UTR contains at least one Kozak sequence. The DNA sequence encoding the target protein is ligated to the coding sequence composed of linked nucleosides to form a whole. A poly-A tail is added to the 3' end of the target DNA sequence. Specifically, the DNA template is synthesized using PCR, and the primer sequences are as follows:

[0028] The linear DNA template was obtained as follows: Using a eukaryotic plasmid encoding luciferase as a template, 1 µg of plasmid DNA was digested in a 10 µL reaction system with BspQ I restriction enzyme (New England Biolabs, catalog number R0712S). Multiple tubes were reacted simultaneously, following the specific reaction system and steps for plasmid digestion:

[0029] Specific reaction procedure: React at 50℃ for 30 min, with the capping temperature set to 75℃. After the reaction, combine the reaction solutions from multiple PCR tubes into a 1.5 mL tube, and then take 4 μL for DNA agarose gel electrophoresis to determine whether the reaction was successful. (Specific agarose gel electrophoresis conditions: 1.5% agarose, constant voltage 180V, 15 min).

[0030] 2. Using a prokaryotic plasmid encoding blue fluorescent protein (BFP) (pRSET-BFP, Yunzhou Biotechnology Guangzhou Co., Ltd., VB160102)

[0031] Using a prokaryotic plasmid encoding green fluorescent protein (sfGFP) (pRSET-sfGFP) (Yunzhou Biotechnology Guangzhou Co., Ltd., VB170101)

[0032] Obtain the DNA template as follows: Use the corresponding DNA polymerase to react in PCR tubes, with a reaction volume of 50 μL per tube. Multiple tubes can be reacted simultaneously, and the DNA template can be amplified according to the specific reaction system below:

[0033] The specific PCR reaction procedure was as follows: pre-denaturation at 95°C for 1 min, denaturation at 95°C for 15 s, annealing at 60°C for 5 s, extension at 72°C for 2 min, for a total of 35 cycles; final extension at 72°C for 5 min. After the reaction, the reaction solutions from multiple PCR tubes were combined into a 1.5 mL tube, and then 4 μL was taken for DNA agarose gel electrophoresis to determine whether the reaction was successful (specific agarose gel electrophoresis conditions: 1.5% agarose, constant voltage 180V, 15 min).

[0034] 3. The reaction product was purified using the HiPure Gel Pure DNA Mini Kit (provided by Guangzhou Meiji Biotechnology, catalog number D2111-03). The purification process was as follows: First, the PCR product was briefly centrifuged, then an equal volume of Buffer GDP was added, and the mixture was vortexed. The HiPure DNA column was placed in the collection tube, and the mixture was transferred to the DNA column and centrifuged at 12000g for 1 min. The filtrate was discarded, and the column was placed back into a 2mL centrifuge tube. 600μL of Buffer DW2 (diluted with anhydrous ethanol) was added to the column, and the column was centrifuged at 12000g for 1 min. The filtrate was discarded, and the column was placed back into a 2mL centrifuge tube. 300μL of Buffer DW2 (diluted with anhydrous ethanol) was added to the column, and the column was centrifuged at 12000g for 2 min. Prepare several nuclease-free 1.5 mL tubes. Place the column back into the 1.5 mL tubes, add 15-30 μL of elution buffer to the center of the column membrane, incubate at room temperature for 2 minutes, centrifuge at 12000g for 1 minute, and then discard the column. Detect and quantify the concentration of template DNA using a Nano Drop Spectrophotometer, recording the 260 / 280 and 260 / 230 ratios. Then store the DNA at -20℃.

[0035] 4. In vitro transcription to synthesize mRNA using chemically modified nucleotides to completely replace natural nucleotides. Base-modified mRNA was synthesized by completely replacing natural nucleotides with chemically modified nucleotides. The synthesis of base-modified mRNA was achieved through in vitro transcription using T7 RNA polymerase. The reaction system for in vitro transcription of prokaryotic BFP mRNA: Taking a 50 μL reaction system as an example, the components shown in the table were added sequentially to a 0.2 mL PCR reaction tube for the reaction.

[0036] The reaction procedure is as follows: Set the thermostat lid to 75°C. Click the thermostat reaction system and incubate at 37°C for 2 hours and 30 minutes. Add 5 μL of DNase XT (RNase-free, provided by New England Biolabs, catalog number M0570S) to each 50 μL prokaryotic mRNA reaction system and incubate at 37°C for 15 minutes to digest the DNA template in the reaction system. The results of transcribing prokaryotic BFP mRNA with triphosphates of different base chemical modifications instead of the corresponding natural nucleoside triphosphates can be seen in the appendix. Figure 2 .

[0037] The reaction system for in vitro transcription of prokaryotic sf GFP mRNA: Taking a 50 μL reaction system as an example, add the components shown in the table to a 0.2 mL PCR reaction tube for the reaction:

[0038] The reaction procedure is as follows: Set the thermostat lid to 75°C. Click the thermostat reaction system and incubate at 37°C for 2 hours and 30 minutes. Add 5 μL of DNase XT (RNase-free, provided by New England Biolabs, catalog number M0570S) to each 50 μL prokaryotic mRNA reaction system and incubate at 37°C for 15 minutes to digest the DNA template in the reaction system. The results of transcribing prokaryotic sfGFP mRNA with different base-modified triphosphates replacing the corresponding types of natural nucleoside triphosphates as substrates can be seen in the appendix. Figure 4 .

[0039] The reaction system for in vitro transcription of eukaryotic mRNA: Taking a 40 μL reaction system as an example, add the components shown in the table to a 0.2 mL PCR reaction tube in sequence for reaction, and replace the natural adenosine triphosphate with the chemically modified adenosine series nucleoside triphosphates in the reaction.

[0040]

[0041] The reaction procedure was as follows: Set the thermostat lid to 75°C. Click the thermostat reaction system and incubate at 37°C for 2 hours and 30 minutes. After incubation, add 4 μL of RNase XI (RNase-free, provided by New England Biolabs, catalog number M0570S) to each 40 μL eukaryotic mRNA reaction system. Then, quench the reacted samples at -80°C for 5 minutes. The results of eukaryotic luciferase mRNA transcribed using chemically modified adenosine triphosphates (with all natural adenosine triphosphates replaced by chemically modified adenosine triphosphates) are shown in the appendix. Figure 6 .

[0042] The reaction system for in vitro transcription of eukaryotic mRNA: Taking a 40 μL reaction system as an example, add the components shown in the table to a 0.2 mL PCR reaction tube in sequence for reaction, and replace the natural cytidine triphosphate with the chemically modified cytidine series nucleoside triphosphates in the reaction.

[0043]

[0044] The reaction procedure was as follows: Set the thermostat lid to 75°C. Click the thermostat reaction system and incubate at 37°C for 2 hours and 30 minutes. After incubation, add 4 μL of RNase XI (RNase-free, provided by New England Biolabs, catalog number M0570S) to each 40 μL eukaryotic mRNA reaction system. Then, quench the reacted samples at -80°C for 5 minutes. The results of eukaryotic luciferase mRNA transcribed using chemically modified cytidine triphosphates (with all natural cytidine triphosphates replaced by chemically modified cytidine series nucleosides) can be seen in the appendix. Figure 8 .

[0045] The reaction system for in vitro transcription of eukaryotic mRNA: Taking a 40 μL reaction system as an example, add the components shown in the table to a 0.2 mL PCR reaction tube in sequence for reaction, and replace the corresponding natural uracil triphosphates with chemically modified uracil series nucleoside triphosphates in the reaction.

[0046]

[0047] The reaction procedure was as follows: Set the thermostat lid to 75°C. Click the thermostat reaction system and incubate at 37°C for 2 hours and 30 minutes. After incubation, add 4 μL of RNase XI (RNase-free, provided by New England Biolabs, catalog number M0570S) to each 40 μL eukaryotic mRNA reaction system. Then, quench the reacted samples at -80°C for 5 minutes. The results of eukaryotic luciferase mRNA transcribed using uracil-based nucleoside triphosphates that completely replaced the corresponding natural uracil triphosphates are shown in the appendix. Figure 11 .

[0048] 5. The prepared chemically modified mRNA was purified according to different purification methods.

[0049] Prokaryotic mRNA purification method: First, suspend the cellulose solution in the elution solution to prepare an elution buffer of 0.2 g cellulose / mL. Place the DNA adsorption column in a 2 mL collection tube (Guangzhou Meiji Biotechnology, DNA / RNA micro-adsorption column, catalog number M021), add 600 μL of the suspension to the DNA column, and vortex for 3 h to activate. Centrifuge the activated cellulose suspension at 14000 g for 1 min, discard the filtrate, and add 500 μL of elution buffer to resuspend the cellulose. Then, transfer the mixture from the in vitro transcription reaction to the DNA column and vortex for 40 min. Centrifuge the DNA column at 14000 g for 1 min, collect the filtrate, and add it to another newly activated DNA column (activation steps as described in step 3), and vortex for 40 min. Centrifuge the DNA column at 14000 g for 1 min, collect the filtrate, and then transfer the filtrate to a new 1.5 mL nuclease-free centrifuge tube pre-chilled on ice. Pre-cool anhydrous isopropanol and 3M sodium acetate solution on ice. Then, add 500 μL of isopropanol and 50 μL of sodium acetate to the centrifuge tube in step (6), gently invert to mix, and centrifuge at 14000g for 10 min at 4°C. Remove the supernatant, add 500 μL of pre-cooled anhydrous ethanol at 4°C to the centrifuge tube, and centrifuge at 14000g for 10 min at 4°C. After centrifugation, remove the supernatant, add 500 μL of pre-cooled anhydrous ethanol at 4°C, and centrifuge at 14000g for 10 min at 4°C. Remove the supernatant, and air-dry the precipitate at room temperature for 30 min. Finally, dissolve the obtained mRNA product in 10-20 μL of nuclease-free water, determine the concentration of purified mRNA using a Nano Drop Spectrophotometer, and simultaneously determine the 260 / 230 and 260 / 280 ratios. Take 500 ng of purified mRNA and perform agarose gel electrophoresis to detect the product. The mRNA solution can be used immediately or stored at -80°C.

[0050] Eukaryotic mRNA purification method: (The Monarch Spin RNAClean up Kits provided by New England Biolabs, catalog number T2040) (1) Add 100µl of Monarch Buffer BX to 50µl of sample. When the sample volume is less than 50µl, adjust the volume to 50µl with nuclease-free water. For samples larger than 50µl, adjust the buffer volume proportionally. (2) Add 150µl of anhydrous ethanol (≥95%) to the sample and mix with a pipette. (3) Place the RNA purification column on the collection tube, transfer the mixture to the purification column and cap it. Centrifuge at 16000×g for 1min, and then discard the filtrate. (4) Put the purification column back on the collection tube, add 500µl of Monarch Buffer WX, centrifuge at 16000×g for 1min, and then discard the filtrate. (5) Repeat the washing process in step 4. (6) Transfer the purification column to a nuclease-free 1.5ml centrifuge tube. (7) Elution was performed using 20 µL of nuclease-free water. The eluted RNA can be used immediately or stored at -80°C. The results of in vitro transcription of chemically modified nucleoside triphosphates are shown in the appendix. Figure 6 , Figure 8 and Figure 10 .

[0051] 6. Expression efficiency of chemically modified mRNA in a cell-free in vitro translation system (1) Expression efficiency of chemically modified mRNA in prokaryotic cell-free in vitro translation system A prokaryotic in vitro translation system was prepared by adding the following components, and the translation of different BFP mRNAs in the PURE in vitro translation system was completed:

[0052] The prepared in vitro translation system was incubated at 37°C for 3 hours. The experimental steps for detecting luminescence using a microplate reader were as follows: the reacted sample was centrifuged at 12000g for 10 minutes at room temperature. Then, 25μL of the corresponding reactant was added to each well of a 96-well plate using a 300μL pipette, and the mixture was thoroughly mixed. Fluorescence intensity was then detected using a microplate reader. The expression efficiency of chemically modified mRNA for BFP protein in the PURE system is shown in the attached figure. Figure 3 As shown. From the appendix Figure 3It was found that the translation efficiency of BFP mRNAs completely replaced by N6-acetyladenine, 7-deadenine, 5-methylcytosine, 5-bromocytosine, α-thiocytosine, 5-methoxyuridine, pseudouridine, methylpseudouridine, and α-thiouridine in the prokaryotic PURE system was higher than that of BFP mRNAs transcribed from natural nucleosides. The degree of enhancement varied among the different types of BFP mRNAs, demonstrating that chemically modified nucleosides can effectively enhance the translation efficiency of transcribed prokaryotic mRNAs in the prokaryotic PURE system to a certain extent.

[0053] (2) A prokaryotic in vitro translation system was prepared by adding the following components, and the translation of different mRNAs in the PURE in vitro translation system was completed:

[0054] The prepared in vitro translation system was incubated at 37°C for 3 hours. The experimental steps for detecting luminescence using a microplate reader are as follows: The reacted sample was centrifuged at 12000g for 10 minutes at room temperature. Then, 25μL of the corresponding reactant was added to each well of a 96-well plate using a 300μL pipette, and the mixture was thoroughly mixed. Fluorescence intensity was then detected using a microplate reader. The expression efficiency of chemically modified mRNA as sfGFP protein in the PURE system is shown in the attached figure. Figure 5 As shown. From the appendix Figure 5 It was found that the translation efficiency of sfGFP mRNA completely replaced by N6-acetyladenine, 5-methylcytosine, 5-bromocytosine, α-thiocytosine, 5-methoxyuracil, pseudouridine, methylpseudouridine, and α-thiouracil in the prokaryotic PURE system was higher than that of sfGFP mRNA transcribed from natural nucleosides. The degree of enhancement varied among the different nucleosides, demonstrating that chemically modified nucleosides can effectively enhance the translation efficiency of transcribed prokaryotic mRNA in the prokaryotic PURE system to a certain extent.

[0055] Example 2 Based on Example 1, this embodiment synthesizes an mRNA encoding luciferase containing a single type of nucleoside modification, detects its expression efficiency in an in vitro translation system of eukaryotic rabbit reticulocyte lysate, translates it into the target protein luciferase, and then uses luciferin as a substrate to oxidize luciferin for chemiluminescence using the translated luciferase. The luminescence intensity is detected using an ELISA reader to determine the translation efficiency of natural and chemically modified mRNAs.

[0056] 1. A eukaryotic in vitro translation system was prepared by adding the following components, and the translation of eukaryotic mRNAs of different chemically modified adenine triphosphates in rabbit reticulocyte lysates was completed:

[0057] The prepared in vitro translation system was incubated at 30°C for 90 min. The experimental steps for detecting luminescence using a microplate reader are as follows: Add 2.5 μL of the protein solution containing luciferase from different samples to a 96-well plate, incubate at room temperature for 10 min, then simultaneously add 50 μL of Luciferase Assay Reagent (Promega, E1483) to each well using a 30-300 μL pipette and mix thoroughly. Detect the corresponding luminescence intensity on the microplate reader within 5 min. The expression rate of chemically modified adenine triphosphate eukaryotic mRNA in the rabbit reticulin lysis buffer system for luciferase protein is shown in the attached figure. Figure 7 As shown.

[0058] From the appendix Figure 7 It was found that the translation efficiency of luciferase mRNAs with complete substitutions of N6-acetyladenine, N6-propionyladenine, N6-butyryladenine, N6-valeryladenine, N6-hexanoyladenine, and 2-aminoadenine in the eukaryotic rabbit reticulin lysis buffer system was higher than that of luciferase mRNAs transcribed from natural nucleosides. Among them, the translation efficiency of mRNAs with complete substitutions of N6-acetyladenine and 2-aminoadenine was the strongest, with an enhancement of nearly 3 times. This proves that chemically modified adenine can effectively enhance the translation efficiency of transcribed luciferase mRNAs in the eukaryotic rabbit reticulin lysis buffer system to a certain extent.

[0059] 2. Prepare a eukaryotic in vitro translation system by adding the following components, and complete the translation of eukaryotic mRNAs with different chemically modified cytosine triphosphates in rabbit reticulocyte lysates:

[0060] The prepared in vitro translation system was incubated at 30°C for 90 min. The experimental steps for detecting luminescence using a microplate reader are as follows: Add 2.5 μL of the protein solution containing luciferase from different samples to a 96-well plate, incubate at room temperature for 10 min, then simultaneously add 50 μL of Luciferase Assay Reagent (Promega, E1483) to each well using a 30-300 μL pipette and mix thoroughly. Detect the corresponding luminescence intensity on the microplate reader within 5 min. The expression rate of luciferase protein in the rabbit reticulin lysis buffer system of chemically modified cytosine triphosphate eukaryotic mRNA is shown in the attached figure. Figure 9 As shown.

[0061] From the appendix Figure 9It was found that the translation efficiency of luciferase mRNAs completely replaced by 5-fluorocytosine, 5-chlorocytosine, 5-bromocytosine, and 5-iodocytosine in the eukaryotic rabbit reticulin lysis buffer system was higher than that of luciferase mRNA transcribed from natural cytosine. Among them, the 5-fluorocytosine-replaced mRNA had the strongest translation efficiency, which was enhanced by about 2.2-2.3 times. This proves that chemically modified cytosine can effectively enhance the translation efficiency of transcribed luciferase mRNA in the eukaryotic rabbit reticulin lysis buffer system to a certain extent.

[0062] 3. Prepare a eukaryotic in vitro translation system by adding the following components, and complete the translation of eukaryotic mRNAs of different chemically modified uracil series nucleoside triphosphates in rabbit reticulocyte lysates:

[0063] The prepared in vitro translation system was incubated at 30°C for 90 min. The experimental steps for detecting luminescence using a microplate reader are as follows: Add 2.5 μL of the protein solution containing luciferase from different samples to a 96-well plate, incubate at room temperature for 10 min, then simultaneously add 50 μL of Luciferase Assay Reagent (Promega, E1483) to each well using a 30-300 μL pipette and mix thoroughly. Detect the corresponding luminescence intensity on the microplate reader within 5 min. The expression rate of luciferase protein in the rabbit reticulin lysis buffer system of chemically modified uracil series nucleoside triphosphate eukaryotic mRNA is shown in the attached figure. Figure 11 As shown.

[0064] From the appendix Figure 11 It was found that the translation efficiency of luciferase mRNAs completely replaced by 5-chlorouracil, 5-bromouracil, 5-iodouracil, 5-methyluracil, 5-methoxyuracil, pseudouridine, and methylpseudouridine in the eukaryotic rabbit reticulin lysis buffer system was higher than that of luciferase mRNA transcribed from natural uracil. Among them, the translation efficiency enhancement of luciferase mRNAs completely replaced by pseudouridine and methylpseudouridine was the most significant, increasing by 5-6 times, while the translation efficiency of luciferase mRNAs completely replaced by 5-chlorouracil and 5-bromouracil was increased by about 2 times. This proves that chemically modified uracil can effectively enhance the translation efficiency of transcribed luciferase mRNAs in the eukaryotic rabbit reticulin lysis buffer system to a certain extent.

[0065] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A base-modified mRNA, characterized in that, The mRNA is transcribed in vitro by completely replacing natural nucleotides with chemically modified nucleotides, and is purified to obtain; The chemically modified nucleotides are obtained by modifying the base and / or sugar of the nucleotide by chemical synthesis or enzymatic synthesis; The chemically modified nucleotides include chemically modified adenine, guanine, cytosine and uracil compounds, salts or isomers thereof.

2. The base-modified mRNA according to claim 1, characterized in that The chemically modified nucleotides are modified by introducing any one or a combination of hydrogen, C2-C5 aliphatic acyl, aromatic acyl, halogenated acyl, C1-C5 alkyl, methoxy, halogen and nitrile group into the base of the nucleotide, and introducing a hydroxyl group, hydrogen, halogen, methoxy, ethoxy or methoxymethyl ether group into the sugar group of the nucleotide.

3. The base-modified mRNA according to claim 2, characterized in that The chemically modified nucleotides are selected from one or more of N1-methyl pseudouridine, phosphorothioate modification, N6-methyladenine, 5-methylcytosine, N4-ethylcytosine, 2-thioguanine and 5-methyluracil.

4. The base-modified mRNA according to claim 1, characterized in that The mRNA comprises at least one or more of the following structural elements: a promoter; or a 5'UTR comprising at least one Kozak sequence, or a 3'UTR, or a coding sequence composed of linked nucleotides, or a poly A tail.

5. The base-modified mRNA according to claim 1, characterized in that The purification process is purified by selective adsorption or high performance liquid chromatography.

6. The use of a base-modified mRNA according to any one of claims 1-5 to improve the translation efficiency of a cell-free protein synthesis system.

7. A method of increasing the translation efficiency of a cell-free protein synthesis system, comprising, The use of a base-modified mRNA according to any one of claims 1-5 in a cell-free protein synthesis process, at least one of Western blot, enzyme-linked immunosorbent assay or radioisotope labeling method is used to detect the translation efficiency, to screen and optimize the mRNA stability and protein expression level, and to establish an optimization strategy for the cell-free protein synthesis system.

8. A cell-free protein synthesis system, characterized by, The system contains a base-modified mRNA according to any one of claims 1-5.

9. The cell-free protein synthesis system of claim 8, wherein, The system is a prokaryotic E. coli PURE system or a eukaryotic system using rabbit reticulocyte lysate as material.

10. The use of a base-modified mRNA according to any one of claims 1-5, or the method for improving the translation efficiency of a cell-free protein synthesis system according to claim 7, or the cell-free protein synthesis system according to claim 8 or 9 in the preparation of vaccine antigens, the construction of artificial biological systems or the synthesis of therapeutic proteins.

Images