Small-amount preparation method of self-replicating RNA (Ribonucleic Acid)

Abstract

The invention provides a small-amount preparation method of self-replicating RNA (Ribonucleic Acid), which comprises the following steps: (1) preparing an IVT reaction template, (2) carrying out IVT reaction, (3) digesting a DNA (Deoxyribonucleic Acid) template, (4) purifying an IVT sample and (5) carrying out capping reaction: adding a capping buffer solution, GTP (Glutathione Triphosphate), SAM (Soluble Amplified Matrix), an RNA enzyme inhibitor, capping enzyme, 2-O-methoxytransferase and denatured RNA into a reaction container, uniformly mixing, and then incubating; and (6) purification: purifying the sample after the capping reaction. According to the method, the in-vitro transcription and capping process of saRNA is optimized, the method is suitable for small-amount preparation of self-replicating RNA, the operation is simple, the self-replicating RNA with high integrity and normal expression can be obtained, and the system can be amplified.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for preparing a small amount of stable saRNA by in vitro transcription and enzymatic reaction of in vitro amplified self-replicating RNA. Background Art

[0002] The basic principle of saRNA technology is to combine viral nonstructural protein genes (such as NSP1-4) with the target gene to form a self-replicating RNA molecule. After entering the host cell, this RNA molecule relies on the catalytic action of RdRp to replicate itself and produce large amounts of target protein mRNA. This technology can significantly reduce the required dosage while improving protein expression levels and immune response efficiency.

[0003] Capping of saRNA (self-amplifying RNA) is one of the key steps in its production process. The main purpose of capping is to improve the stability and translation efficiency of RNA while reducing immunogenicity, thereby enhancing the effect of vaccines or therapeutic drugs. There are two ways to cap mRNA, enzymatic capping and co-transcriptional capping. Enzymatic capping has a high capping efficiency, which can reach close to 100%, but the operation is complicated and the cost is high. saRNA usually requires a 5'Cap 1 structure to ensure its stability and translation efficiency in the cell. The Cap 1 structure is formed by the GpppN core plus methylation modification, which is the main cap structure in higher organisms. Different capping structures may affect the immunogenicity and efficacy of saRNA. For example, uncapped RNA or pseudo-symmetric cap structures may trigger an antiviral response, thereby reducing vaccine efficacy.

[0004] Currently, in vitro transcription and enzymatic capping processes are mainly used for conventional mRNA molecules. However, due to the large molecular weight and more complicated operation process of self-replicating saRNA, existing reagents and processes are difficult to apply to saRNA. Therefore, it is crucial to develop a small-scale preparation method for saRNA that is simple to operate, has high integrity and capping efficiency for the saRNA process. Summary of the Invention

[0005] The purpose of the present invention is to provide a saRNA preparation method with simple operation and high capping efficiency based on the existing technology. This method is particularly suitable for the preparation of saRNA with a base number of 8000nt-15000nt.

[0006] The purpose of the present invention can be achieved by the following measures:

[0007] A method for preparing a small amount of self-replicating RNA comprises the following steps:

[0008] (1) Preparation of IVT reaction template: linearized plasmid was used as the IVT reaction template;

[0009] (2) IVT reaction: dilution water, in vitro transcription buffer, nucleoside triphosphates, RNA inhibitor, inorganic pyrophosphatase, T7 RNA polymerase, and linearized template were added to the reaction vessel, mixed, and incubated;

[0010] (3) DNA template digestion: After the IVT reaction is completed, deoxyribonuclease I is added, mixed, and incubated to digest the DNA template;

[0011] (4) IVT sample purification: Purify the sample after IVT reaction using lithium chloride;

[0012] (5) Capping reaction: Add capping buffer, GTP, SAM, RNase inhibitor, capping enzyme, 2'-O-methoxytransferase, and RNA to the reaction vessel, mix well, and then incubate;

[0013] (6) Purification: Purify the sample after the capping reaction using lithium chloride or affinity chromatography.

[0014] The saRNA-IVT sample to be capped in the present invention can be prepared by completing the in vitro transcription step of the saRNA linearized plasmid according to the in vitro transcription step, and then purifying it.

[0015] The capping method of the present invention is suitable for various saRNAs with a base number of 8000nt-15000nt. As long as the base type meets the requirements, there is no limitation on the specific saRNA. In a preferred embodiment, saRNA-IVT can include luci-IVT-saRNA.

[0016] In step (1), the linearized template is prepared by completing the saRNA linearized plasmid according to the plasmid linearization reaction step and then purified. For example, the linearized plasmid can be a linearized plasmid obtained by digesting the saRNA plasmid with BspQI. Taking the luciferase-saRNA plasmid as an example, it is first digested with BspQI to obtain a linearized plasmid, and then purified to obtain the luciferase-saRNA linearized product Linearized plasmid template.

[0017] In step (2), the concentration range of nucleoside triphosphates is 0.5mmol / ul~1mmol / ul, the concentration range of inorganic pyrophosphatase is 0.001U / ul~0.005U / ul, the concentration range of T7 RNA polymerase is 1U / μl~10U / μl, the concentration range of RNA inhibitor is 1U / / μl~2U / μl, and the concentration range of linearized template is 0.0lug / u1~0.lug / ul; nucleoside triphosphates include GTP, CTP, ATP and UTP.

[0018] In step (2), the incubation temperature is 37° C., and the incubation time is 90 min to 150 min.

[0019] In step (3), the concentration of deoxyribonuclease I ranges from 0.05 U / μL to 0.5 U / uL.

[0020] In step (3), the incubation temperature is 37° C., and the incubation time is 10 min to 30 min.

[0021] In step (4), the conditions for purification using lithium chloride are as follows: saRNA is precipitated with lithium chloride at a final concentration of 2.5 mol, and low-temperature precipitation is performed at least twice.

[0022] In step (5), preferably, the amount of the denatured saRNA-IVT template has a preferred template concentration of 1 μg / μl to 70 μg / μl.

[0023] In step (5), preferably, the concentration of GTP is 0.2 mmol / μl to 1 mmol / μl, the concentration of SAM is 0.2 mmol / μl to 2 mmol / μl, the concentration of RNase inhibitor is 1 U / / μl to 2 U / μl, the concentration of capping enzyme is 0.1 U / μl to 1 U / μl, and the concentration of 2'-O-methoxytransferase is 1 U / μl to 5 U / μl.

[0024] In step (5), preferably, the incubation temperature is 37° C. and the incubation time is 40 min.

[0025] In step (6), preferably, lithium chloride can be used to purify the sample after the capping reaction. The purification conditions using lithium chloride are as follows: saRNA is precipitated with lithium chloride at a final concentration of 2.5 mol, the precipitation temperature is -20°C, and the precipitation number is at least 2 times.

[0026] In step (6), as an option, the integrity and cellular expression of the purified sample can also be detected, such as the obtained integrity map, the saRNA integrity calculated as the peak area percentage, the ELISA detection cell expression result map, etc.

[0027] The in vitro transcription buffer in the present invention may also be referred to as an in vitro transcription buffer.

[0028] The nucleoside triphosphate (NTP) in the present invention is also known as nucleoside triphosphate, and common forms in nature include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) or uridine triphosphate (UTP).

[0029] Deoxyribonuclease I in the present invention may also be referred to as DNaseI.

[0030] GTP in the present invention refers to guanosine triphosphate, that is, guanine-5'-triphosphate, and its full name in biochemistry is 9-β-D-ribofuranoseguanine-5'-triphosphate, or 9-β-D-ribofuranose-2-amino-6-oxy-purine-5'-triphosphate.

[0031] SAM in the present invention refers to S-adenosylmethionine, which is a sulfur-containing primary metabolite widely present in prokaryotic and eukaryotic cells.

[0032] The RNAse inhibitors in the present invention include RNase inhibitors.

[0033] The capping enzymes of the present invention include vaccinia capping enzymes.

[0034] The capping buffer in the present invention includes an annexin binding buffer, preferably a 10X capping reaction buffer.

[0035] The reagents used in the present invention are preferably reagents from specific manufacturers and combinations thereof.

[0036] The saRNA preparation method of the present invention has multiple technical advantages for saRNA with a large number of bases and poor stability.

[0037] (1) The saRNA small-scale preparation method provided by the present invention can quickly complete the small-scale preparation of long-fragment saRNA. The process operation is simple and fast, and meets the process requirements.

[0038] (2) The saRNA small-scale preparation method provided by the present invention can produce 70 μg / μl saRNA in a single reaction, with an integrity of more than 70%. It has high overall yield, good integrity, and high cellular expression, and has obvious technical advantages.

[0039] (3) The small-scale preparation method of saRNA provided by the present invention is simple, highly operable, and the process is easy to scale up. It has strong technical advantages for the in vitro transcription process of long-fragment concentration saRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1is the capillary gel electrophoresis pattern of the RNA ladder microfluidic chip in Example 1 of the present invention;

[0041] Figure 2 This is the capillary gel electrophoresis pattern of the microfluidic chip after capping of saRNA-1 in Example 1 of the present invention;

[0042] Figure 3 This is the saRNA-1 cell protein expression detection result in Example 1 of the present invention;

[0043] Figure 4 This is the capillary gel electrophoresis pattern of the microfluidic chip sample after capping of saRNA-2 in Example 2 of the present invention;

[0044] Figure 5 This is the saRNA-2 cell protein expression detection result in Example 2 of the present invention;

[0045] Figure 6 This is the capillary gel electrophoresis pattern of the microfluidic chip sample after luciferase-saRNA capping in Example 3 of the present invention;

[0046] Figure 7 This is the luciferase protein expression detection result in Example 3 of the present invention. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0048] Example 1. Experimental process for preparing saRNA-1 samples

[0049] 1.1 Initial reaction template preparation

[0050] saRNA linearized plasmid sample: The saRNA-1 plasmid was digested with BspQI to obtain a linearized plasmid sample. This is only an example. The method of the present invention is applicable to any saRNA plasmid with a length of 8000 nt-15000 nt.

[0051] In vitro transcription reaction reagents: T7 polymerase, inorganic pyrophosphatase, in vitro transcription buffer, NTP, and RNA inhibitor were obtained from Hanhai New Enzyme Biotechnology Co., Ltd., Jinan Protein Co., Ltd., or Yisheng Biotechnology (Shanghai) Co., Ltd.

[0052] IVT-saRNA sample diluent: sterile enzyme-free water;

[0053] 1.2 In vitro transcription reaction

[0054] In a 1.5 mL enzyme-free centrifuge tube, add the following reagents in corresponding volumes, mix well, and place in a metal bath heated to 37°C for 120 min.

[0055]

[0056] 1.3 DNA template digestion

[0057] Add 1 μl of DNase I to the above reaction solution and incubate at 37°C for 15 min.

[0058] 1.4 Post-digestion sample purification

[0059] Add 12u1 of 7.5M lithium chloride to the digested EP tube, mix well, centrifuge briefly for 30s, place in a -20°C refrigerator for 30min, then centrifuge at 8000rmp for 15min, collect the precipitate, wash the precipitate with 1ml of 70% ethanol, repeat the above operation once, and finally dissolve the precipitate with 100u1 of enzyme-free water.

[0060] Capping reaction reagents: capping enzyme, 2-O-methyltransferase, capping buffer, GTP, SAM, and RNA inhibitor were obtained from Hanhai New Enzyme Biotechnology Co., Ltd., Jinan Protein Co., Ltd., or Yisheng Biotechnology (Shanghai) Co., Ltd.

[0061] 1.5saRNA capping reaction

[0062] In an enzyme-free 1.5 ml centrifuge tube, add the corresponding volume of solution in sequence, mix well, place it in a metal bath that has been heated to 37°C, and incubate for 30 minutes.

[0063]

[0064]

[0065] 1.6 Sample purification after capping

[0066] Add 12 μl of 7.5 M lithium chloride to the capped EP tube, mix well, and place in a -20°C refrigerator for 30 min. Then, centrifuge at 8000 rpm for 15 min, collect the precipitate, and wash it with 1 ml of 70% ethanol. Repeat the above steps once, and finally dissolve it with 30 μl of enzyme-free water.

[0067] 1.7 Sample testing

[0068] Concentration detection: Nanodrop was used to detect the concentration of saRNA samples.

[0069] Integrity detection: PE Labchip was used to detect the integrity of the collected saRNA.

[0070] Cell expression detection: Elisa was used to detect the expression of saRNA.

[0071] 1.8 Experimental Results

[0072] (1) The average concentration of purified saRNA detected by Nanodrop was 2050 ng / μl.

[0073] (2) Figure 1 It is the RNA Ladder integrity map; Figure 2 This is the integrity map of the saRNA-1 sample, with an integrity of 70.9%; Figure 3 This is the result of Elisa detection of saRNA-1 cell expression.

[0074] Example 2. Experimental process for preparing saRNA-2 samples

[0075] 2.1 Initial reaction template preparation

[0076] saRNA linearized plasmid sample: saRNA-2 plasmid was digested with BspQI to obtain a linearized plasmid sample. This is only an example. The method of the present invention is applicable to any saRNA plasmid with a length of 8000 nt-15000 nt.

[0077] In vitro transcription reaction reagents: T7 polymerase, inorganic pyrophosphatase, in vitro transcription buffer, NTP, and RNA inhibitor were obtained from Hanhai New Enzyme Biotechnology Co., Ltd., Jinan Protein Co., Ltd., or Yisheng Biotechnology (Shanghai) Co., Ltd.

[0078] IVT-saRNA sample diluent: sterile enzyme-free water;

[0079] 2.2 In vitro transcription reaction

[0080] In a 1.5 ml enzyme-free centrifuge tube, add the following reagents in corresponding volumes in sequence, mix well, place the tube in a metal bath heated to 37°C, and incubate for 120 minutes.

[0081]

[0082] 2.3 DNA template digestion

[0083] Add 1 μl of DNase I to the above reaction solution and incubate at 37°C for 15 min.

[0084] 2.4 Post-digestion sample purification

[0085] Add 12u1 of 7.5M lithium chloride to the digested EP tube, mix well, centrifuge briefly for 30s, place in a -20°C refrigerator for 30min, then centrifuge at 8000rmp for 15min, collect the precipitate, wash the precipitate with 1ml of 70% ethanol, repeat the above operation once, and finally dissolve the precipitate with 100u1 of enzyme-free water.

[0086] Capping reaction reagents: capping enzyme, 2-O-methyltransferase, capping buffer, GTP, SAM, and RNA inhibitor were obtained from Hanhai New Enzyme Biotechnology Co., Ltd., Jinan Protein Co., Ltd., or Yisheng Biotechnology (Shanghai) Co., Ltd.

[0087] 2.5 saRNA capping reaction

[0088] In an enzyme-free 1.5 ml centrifuge tube, add the corresponding volume of solution in sequence, mix well, place it in a metal bath that has been heated to 37°C, and incubate for 30 minutes.

[0089] Serial number Reagent name Concentrations of various reagents Sample volume 1 10X capping reaction buffer 1X 2 μl 2 SAM (20mM) 1.2mmol / μl 0.5 μl 3 <![CDATA[IVT -sa RNA-2 Template]]> 5 μg / μl 13 μl 4 GTP (10 mM) 1mmol / μl 2 μl 5 Vaccinia capping enzyme (10 U / μl) 1U / μl 1 μl 6 2'-O-methyltransferase (50 U / μl) 2U / μl 1 μl 7 RNase inhibitor (40 U / μl) 1U / / μl 0.5 μl

[0090] 2.6 Sample purification after capping

[0091] Add 12 μl of 7.5 M lithium chloride to the capped EP tube, mix well, and place in a -20°C refrigerator for 30 min. Then, centrifuge at 8000 rpm for 15 min, collect the precipitate, and wash it with 1 ml of 70% ethanol. Repeat the above steps once, and finally dissolve it with 30 μl of enzyme-free water.

[0092] 2.7 Sample testing

[0093] Concentration detection: Nanodrop was used to detect the concentration of saRNA samples.

[0094] Integrity detection: PE Labchip was used to detect the integrity of the collected saRNA.

[0095] Cell expression detection: Elisa was used to detect the expression of saRNA.

[0096] 2.8 Experimental Results

[0097] (1) The average concentration of purified saRNA detected by Nanodrop was 1400 ng / μl.

[0098] (2) Figure 4 This is the integrity map of the saRNA-2 sample, with an integrity of 66.8%; Figure 5 This is the result of Elisa detection of cell expression after saRNA-2 capping.

[0099] Example 3. Experimental process for preparing luciferase-saRNA samples

[0100] 3.1 Initial reaction template preparation

[0101] saRNA linearized plasmid sample: The luciferase-saRNA plasmid was digested with BspQI to obtain a linearized plasmid sample. This is only an example. The method of the present invention is applicable to any saRNA plasmid with a length of 8000nt-15000nt.

[0102] In vitro transcription reaction reagents: T7 polymerase, inorganic pyrophosphatase, in vitro transcription buffer, NTP, and RNA inhibitor were obtained from Hanhai New Enzyme Biotechnology Co., Ltd., Jinan Protein Co., Ltd., or Yisheng Biotechnology (Shanghai) Co., Ltd.

[0103] IVT-saRNA sample diluent: sterile enzyme-free water;

[0104] 3.2 In vitro transcription reaction

[0105] In a 1.5 mL enzyme-free centrifuge tube, add the following reagents in corresponding volumes, mix well, and place in a metal bath heated to 37°C for 120 min.

[0106]

[0107] 3.3 DNA template digestion

[0108] Add 1 μl of DNase I to the above reaction solution and incubate at 37°C for 15 min.

[0109] 3.4 Post-digestion sample purification

[0110] Add 12u1 of 7.5M lithium chloride to the digested EP tube, mix well, centrifuge briefly for 30s, place in a -20°C refrigerator for 30min, then centrifuge at 8000rmp for 15min, collect the precipitate, wash the precipitate with 1ml of 70% ethanol, repeat the above operation once, and finally dissolve the precipitate with 100u1 of enzyme-free water.

[0111] Capping reaction reagents: capping enzyme, 2-O-methyltransferase, capping buffer, GTP, SAM, and RNA inhibitor were obtained from Hanhai New Enzyme Biotechnology Co., Ltd., Jinan Protein Co., Ltd., or Yisheng Biotechnology (Shanghai) Co., Ltd.

[0112] 3.5saRNA capping reaction

[0113] In an enzyme-free 1.5 ml centrifuge tube, add the corresponding volume of solution in sequence, mix well, place it in a metal bath that has been heated to 37°C, and incubate for 30 minutes.

[0114] Serial number Reagent name Concentrations of various reagents Sample volume 1 10X capping reaction buffer 1X 2 μl 2 SAM (20mM) 1.2mmol / μl 0.5 μl 3 luciferase-IVT-saRNA template 5 μg / μl 13 μl 4 GTP (10 mM) 1mmol / μl 2 μl 5 Vaccinia capping enzyme (10 U / μl) 1U / μl 1 μl 6 2'-O-methyltransferase (50 U / μl) 2U / μl 1 μl 7 RNase inhibitor (40 U / μl) 1U / / μl 0.5 μl

[0115] 3.6 Sample purification after capping

[0116] Add 12 μl of 7.5 M lithium chloride to the capped EP tube, mix well, and place in a -20°C refrigerator for 30 min. Then, centrifuge at 8000 rpm for 15 min, collect the precipitate, and wash it with 1 ml of 70% ethanol. Repeat the above steps once, and finally dissolve it with 30 μl of enzyme-free water.

[0117] 3.7 Sample testing

[0118] Concentration detection: Nanodrop was used to detect the concentration of saRNA samples.

[0119] Integrity detection: PE Labchip was used to detect the integrity of the collected saRNA.

[0120] Cell expression detection: Detect the expression of luciferase.

[0121] 3.8 Experimental Results

[0122] (1) The average concentration of purified saRNA detected by Nanodrop was 1800 ng / μl.

[0123] (2) Figure 6 This is the integrity map of the luciferase-saRNA sample, with an integrity of 71%; Figure 7 This is the result of luciferase expression detection after luciferase-saRNA capping.

Claims

1. A method for preparing a small amount of self-replicating RNA, characterized in that: The following steps are involved: (1) Preparation of IVT reaction template: Use linearized plasmid as IVT reaction template; (2) IVT reaction: dilution water, in vitro transcription buffer, nucleoside triphosphates, RNA inhibitor, inorganic pyrophosphatase, T7 RNA polymerase, and linearized template were added to the reaction vessel, mixed, and incubated; (3) DNA template digestion: After the IVT reaction is completed, deoxyribonuclease I is added, mixed, and incubated to digest the DNA template; (4) IVT sample purification: Purify the sample after IVT reaction using lithium chloride; (5) Capping reaction: Add capping buffer, GTP, SAM, RNase inhibitor, capping enzyme, 2´-O-methoxytransferase, and RNA to the reaction vessel, mix well, and then incubate; (6) Purification: Purify the sample after capping reaction using lithium chloride or affinity chromatography.

2. The method according to claim 1, characterized in that In step (1), the linearized plasmid is a linearized plasmid obtained by digesting the saRNA plasmid with BspQI, and the number of bases of the saRNA plasmid is 8000nt to 15000nt.

3. The method according to claim 1, characterized in that In step (2), the concentration range of nucleoside triphosphate is 0.5 mmol / μl to 1 mmol / μl, the concentration range of inorganic pyrophosphatase is 0.001 U / μl to 0.005 U / μl, the concentration range of T7 RNA polymerase is 1 U / μl to 10 U / μl, the concentration range of RNA inhibitor is 1 U / μl to 2 U / μl, and the concentration range of linearized template is 0.01 μg / μl to 0.1 μg / μl.

4. The method according to claim 1, wherein In step (2), the incubation temperature is 37° C. and the incubation time is 90 min to 150 min.

5. The method according to claim 1, characterized in that In step (3), the concentration of deoxyribonuclease I ranges from 0.05 U / μL to 0.5 U / μL, the incubation temperature is 37° C., and the incubation time is 10 min to 30 min.

6. The method according to claim 1, characterized in that In step (4), the conditions for purification using lithium chloride are as follows: saRNA is precipitated with lithium chloride at a final concentration of 2.5 mol, the precipitation temperature is -20°C, and the precipitation times are at least 2 times.

7. The method according to claim 1, characterized in that In step (5), the concentration of GTP is 0.2 mmol / μl to 1 mmol / μl, the concentration of SAM is 0.2 mmol / μl to 2 mmol / μl, the concentration of RNase inhibitor is 1 U / μl to 2 U / μl, the concentration of capping enzyme is 0.1 U / μl to 1 U / μl, the concentration of 2´-O-methoxytransferase is 1 U / μl to 5 U / μl, and the concentration of the denatured saRNA-IVT sample is 1 μg / μl to 70 μg / μl.

8. The method according to claim 1, characterized in that In step (5), the incubation temperature is 37° C. and the incubation time is 40 min; the capping enzyme is vaccinia capping enzyme.

9. The method according to claim 1, characterized in that In step (6), lithium chloride is used to purify the sample after the capping reaction. The purification conditions are: saRNA is precipitated with lithium chloride at a final concentration of 2.5 mol, the precipitation temperature is -20°C, and the precipitation times are at least 2 times.

10. The method according to claim 1, characterized in that Step (6) also includes testing the integrity and cellular expression of the purified sample, including an integrity profile, saRNA integrity calculated as peak area percentage, and cellular expression test results.

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