Method for simply and efficiently preparing circRNA by one-pot method

Abstract

The invention belongs to the technical field of biology. More specifically, the invention relates to a method for simply and efficiently preparing circRNA by a one-pot method. In the same reaction container, reacting the circRNA in-vitro transcription reaction system for 1-4 hours at 37-50 DEG C, and then reacting for 0.5-1.5 hours at 50-65 DEG C; and then directly and sequentially adding DNA enzyme and RNA enzyme for treatment, and purifying to obtain circRNA. According to the method, the preparation efficiency of circRNA is remarkably improved. The method is also matched with a special RNase R buffer solution, and linear RNA impurities can be directly degraded in a reaction system. According to the preparation method, a plurality of key steps such as in-vitro transcription, self-splicing cyclization, template DNA removal and linear RNA impurity degradation can be integrated in a single reaction tube to be continuously completed, the technological process is highly integrated, the efficiency is remarkably improved, and the pollution risk is reduced. According to the method, a staged temperature control strategy is adopted, so that the cyclization efficiency stably exceeds 90%, the reaction time is shortened to several hours, the industrial production value is high, and the method has good application prospect and value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. More specifically, it relates to a simple and efficient one-pot method for preparing circRNA. Background Technology

[0002] Circular RNAs (circRNAs) are a class of single-stranded, closed circular RNA molecules. They are formed by linking the 3' and 5' ends of pre-mRNA through exon or intron circularization, creating a closed circular structure. This structure lacks 5'-Cap and 3'-polyA, and has no free ends, allowing circRNAs to avoid degradation by exonucleases and exhibiting higher stability than linear mRNAs. Therefore, circRNAs have attracted widespread attention due to their stable closed-circular structure, persistent high-level protein expression, and low immunogenicity. They have shown revolutionary application prospects, particularly in gene regulation, protein coding, and disease treatment, especially in vaccine development and biologics. Based on this, research on circRNAs has experienced explosive growth in recent years. However, many challenges remain to be overcome before they can be developed into a mature next-generation RNA drug development platform, especially in areas such as circRNA sequence and structure optimization, in vitro synthesis processes, purification techniques, and delivery systems. Among these, in vitro synthesis processes are considered a key bottleneck currently limiting the development of circRNA technology.

[0003] Currently, the main in vitro synthesis methods for circRNA include chemical ligation, T4 ligase method, and ribozyme method. In comparison, the ribozyme method is simpler to operate, has higher efficiency in circularizing long RNA fragments, and is easier to scale up, thus becoming the most mainstream synthesis strategy in current research. Common ribozyme methods are mainly divided into two strategies: "one-step method" and "two-step method". The basic processes of the two are similar. The one-step method usually includes the following steps: (1) in vitro transcription (IVT); (2) RNA circularization; (3) DNase I treatment to remove template DNA; (4) purification of circRNA stock solution to remove impurities such as enzyme protein and salt ions; (5) RNase R treatment to remove linear RNA impurities; (6) product purification. The two-step method adds an RNA purification step after IVT, and then performs RNA circularization. The two-step method has higher cyclization efficiency because the IVT product is purified before cyclization, but this process leads to greater RNA loss and increases costs. The one-step method eliminates the intermediate purification step, resulting in lower overall costs and higher RNA recovery, but the IVT reaction time is longer (including the time required for cyclization) and the cyclization efficiency is relatively lower.

[0004] More importantly, regardless of whether it's a one-step or two-step method, existing preparation processes cannot complete all operations in a single reaction system. Steps such as DNase I treatment, RNase R degradation, and purification all require changing different reaction tubes, making the operation cumbersome. This not only increases the risk of loss and contamination due to human intervention but also reduces overall efficiency. In the process of large-scale industrial production of circRNA, this multi-step transfer method will significantly increase raw material and time costs, while also affecting the final yield and consistency.

[0005] Therefore, there is an urgent need to develop a preparation technology that can efficiently produce circRNA and complete all key steps in the same reaction system, so as to simplify the operation process, reduce losses, and achieve efficient and large-scale production of circRNA. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of existing circRNA preparation methods, such as low circRNA production efficiency, cumbersome steps, and low circularization efficiency, and to provide a one-pot method for preparing circRNA that is efficient, stable, high-purity, easy to operate, and low-cost.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: The in vitro synthesis of circRNA in this invention employs a type I intron self-splicing system using ribozyme technology, namely the most commonly used in vitro PIE system ligation method. Precursor RNA is synthesized via in vitro transcription, and then undergoes two transesterification reactions in the presence of magnesium ions and GTP to form circRNA.

[0008] This invention provides a method for preparing circRNA, comprising the following steps: (1) The in vitro transcription reaction system of circRNA was subjected to a two-stage reaction: The circRNA in vitro transcription reaction system was subjected to the first stage reaction at 37–50°C for 1–4 h; then the second stage reaction was carried out at 50–65°C for 0.5–1.5 h. (2) Enzyme treatment: The reaction products were sequentially treated with DNA enzyme and RNA enzyme; (3) Purify to obtain circRNA; The two-stage reaction, enzyme treatment, and purification are all completed in the same reaction vessel.

[0009] Preferably, the temperature of the first stage of in vitro transcription reaction is 40~45℃, and the reaction time is 2~4 hours.

[0010] More preferably, the temperature of the first stage of in vitro transcription reaction is 40°C, and the reaction time is 2 to 4 hours.

[0011] More preferably, the temperature of the first stage of in vitro transcription reaction is 40°C and the reaction time is 2 hours.

[0012] Preferably, the temperature of the second-stage in vitro transcription reaction is 50-55°C, and the reaction time is 0.5-1 hour.

[0013] More preferably, the temperature of the second-stage in vitro transcription reaction is 55°C, and the reaction time is 1 hour.

[0014] As an alternative implementation, the RNase treatment is performed by adding RNase buffer and RNase to the reaction system; the RNase buffer is any one of a mixed solution of MgCl2 and KCl, a mixed solution of MgCl2 and LiCl, a MgCl2 solution, or a LiCl solution.

[0015] As an alternative implementation, the final concentration of inorganic salts in the RNase buffer is 1-10 M.

[0016] As an alternative implementation, the final concentration of inorganic salts in the RNase buffer is 1-3 M.

[0017] As an alternative implementation, the final concentration of inorganic salts in the RNase buffer is 2 M.

[0018] Preferably, the RNase buffer is a 1-3 mol / L lithium chloride solution or a magnesium chloride solution.

[0019] Preferably, the RNase buffer is a 2 mol / L lithium chloride solution or a magnesium chloride solution.

[0020] Optionally, the RNase is ribonuclease R.

[0021] As an alternative implementation, the ratio of the added RNase buffer volume to the volume of the in vitro transcription reaction system is (0.5~1.5):10 (preferably 1:10).

[0022] As an alternative implementation, the RNase treatment conditions are: treatment at 35-39°C for 20-40 minutes (preferably incubation at 37°C for 30 minutes).

[0023] As an alternative implementation, the DNase treatment involves adding DNase to the in vitro transcription reaction system and incubating at 35–39°C for 20–40 minutes (preferably at 37°C for 30 minutes) to digest residual linear plasmid template.

[0024] Optionally, the DNA enzyme is deoxyribonuclease I (DNase I).

[0025] As an alternative implementation, the in vitro transcription reaction system comprises the following components: T7 RNA polymerase transcription buffer, UTP, ATP, CTP, GTP, T7 RNA polymerase, inorganic pyrophosphatase, linear plasmid template, and water.

[0026] As an alternative implementation, the final concentrations of UTP, ATP, and CTP are 5 mM.

[0027] Preferably, the final concentration of GTP in the in vitro transcription reaction system is 12.5~17.5mM.

[0028] More preferably, the final concentration of GTP in the in vitro transcription reaction system is 12.5~15mM.

[0029] More preferably, the final concentration of GTP in the in vitro transcription reaction system is 15 mM.

[0030] Preferably, the final concentration of T7 RNA polymerase in the in vitro transcription reaction system is 5~12.5 U / μL.

[0031] As an alternative implementation, the final concentration of T7 RNA polymerase in the in vitro transcription reaction system is 10 U / μL.

[0032] The present invention has the following beneficial effects: 1. This invention employs a staged temperature-controlled in vitro transcription strategy, which enables the circularization efficiency of circRNA to be stably maintained at over 90%, while significantly reducing the total reaction time to within a few hours. This method has great potential for large-scale industrial production of circRNA, effectively reducing unit production costs and meeting the future demand for large quantities of circRNA drugs.

[0033] 2. This invention provides a simple and efficient one-pot method for preparing circRNA, with a highly integrated process flow and convenient and efficient operation. According to the preparation method of this invention, multiple key steps, including in vitro transcription (IVT), self-splicing and circularization, template DNA removal (DNase I treatment), and linear RNA impurity degradation (RNase R digestion), can be continuously completed in a single reaction tube. This "one-tube" operation mode completely eliminates the intermediate purification, transfer, and multiple tube opening steps required in traditional "two-step" or conventional "one-step" methods, greatly simplifying the experimental process and significantly reducing RNA loss, contamination risks, and time costs caused by manual operation and sample transfer.

[0034] 3. This invention utilizes a specially formulated RNase R buffer (Rbuffer), which allows for efficient activation of RNase R directly in the reaction system without purifying the IVT stock solution. This specifically degrades residual linear RNA precursors and transcription byproducts without affecting the stability of the target circRNA. Combined with subsequent affinity purification systems (such as oligo dT purification), high-purity circRNA final products can be obtained. Capillary electrophoresis analysis shows that its integrity is over 97%, ensuring high product quality.

[0035] 4. The circRNA prepared by the method of this invention has a complete closed-circle structure and exhibits excellent biological functions. Cell transfection experiments show that this circRNA can achieve high-level and persistent protein expression in cells. Even on day 7 after transfection, the positive rate of protein expression can still be maintained at about 80%, which fully demonstrates its good translation ability and intracellular stability.

[0036] In summary, this invention provides a highly integrated, simple, and efficient method for preparing circRNA, solving the key problems of cumbersome processes, long processing times, and high losses in existing technologies, and providing strong technical support for the basic research and industrial application of circRNA. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the connection of a type I intron self-splicing system (linearized plasmid template).

[0038] Figure 2 The results of the two-step preparation of circRNA are shown in the figure (M: DL5000 Marker, lane 1: circRNA precursor; lane 2: circRNA after cyclization reaction).

[0039] Figure 3 The results of the one-step preparation of circRNA are shown in the figure (M: DL5000 Marker; 3.5: circRNA sample after IVT reaction for 3.5 h; 16: circRNA sample after IVT reaction for 16 h).

[0040] Figure 4 The results of the effect of GTP on circularization efficiency in the one-step method are shown in the figure (M: DL5000 Marker; Lane 1: circRNA sample obtained by IVT when the amount of GTP is increased to 15mM).

[0041] Figure 5 The results of the effect of GTP and MgCl2 dosage on cyclization efficiency in the one-step method are shown in the figure (M: DL5000 Marker; 1, 2, 3, 4, 5: electrophoresis results of different groups).

[0042] Figure 6 The results of the effect of thermostable T7 RNA polymerase on cyclization efficiency are shown in the figure (M: DL5000 Marker; 1, 2: replicate group, samples with IVT reaction temperature of 50℃; 3: sample with IVT reaction temperature of 55℃).

[0043] Figure 7 The results show the effect of in vitro transcription time on circularization efficiency in the one-step method (M: DL5000 Marker; 1, 2: circRNA samples with IVT reaction time of 6 h; 3, 4: circRNA samples with IVT reaction time of 4 h).

[0044] Figure 8 The results show the effect of in vitro transcription temperature on circularization efficiency in the one-step method (M: DL5000 Marker; 1: circRNA sample of IVT reacted at 40℃ for 16h).

[0045] Figure 9 The results show the effect of in vitro transcription conditions on cyclization efficiency in the one-pot method (M: DL5000 Marker; number: group under different reaction conditions).

[0046] Figure 10 The effect of T7 RNA polymerase dosage on cyclization efficiency in the one-pot method is shown in the figure (M: DL5000 Marker; the numbers correspond to treatment groups 1 to 4 respectively).

[0047] Figure 11 The results of the effect of the reaction temperature in the first stage of the one-pot reaction on the cyclization efficiency are shown in the figure (1: sample with a reaction temperature of 37℃; 2: sample with a reaction temperature of 40℃; 3: sample with a reaction temperature of 45℃; 4: sample with a reaction temperature of 50℃).

[0048] Figure 12 The result is a graph showing the effect of the first stage reaction time on the cyclization efficiency in the one-pot process (M: DL5000 Marker; 1: Sample with a reaction time of 2 h; 2: Sample with a reaction time of 3 h; 3: Sample with a reaction time of 4 h).

[0049] Figure 13 The results show the effect of the reaction temperature in the second stage of the one-pot reaction on the cyclization efficiency (1: sample with a reaction temperature of 50℃; 2: sample with a reaction temperature of 55℃; 3: sample with a reaction temperature of 60℃; 4: sample with a reaction temperature of 65℃).

[0050] Figure 14The results show the effect of the second stage reaction time on the cyclization efficiency in the one-pot process (1: sample with a reaction time of 0.5 h; 2: sample with a reaction time of 1 h; 3: sample with a reaction time of 1.5 h).

[0051] Figure 15 The results of the effect of GTP dosage on cyclization efficiency in the one-pot process are shown in the figure (M: DL5000 Marker; numbers 1 to 4 correspond to final GTP concentrations of 12.5 mM, 15 mM, 17.5 mM, and 20 mM, respectively).

[0052] Figure 16 The results show the effect of Rbuffer on the efficiency of RNaseR digestion of linear RNA in the one-pot method (M: DL5000 Marker; the numbers correspond to different groups).

[0053] Figure 17 The effect of the amount of Rbuffer used in the one-pot digestion method on the efficiency of RNaseR digestion of linear RNA is shown in the figure (M: DL5000 Marker).

[0054] Figure 18 This is a chromatographic diagram of the purification of circular RNA.

[0055] Figure 19 Agarose gel electrophoresis image; M: DL5000 Marker; 1: Circular RNA (untreated with RNaseR) sample; 2: Circular RNA (treated with RNaseR) sample; 3: Loading solution sample; 4: 1A-1 sample (through-flow peak); 5: 1A-2 sample (through-flow peak); 6: 1A-3 sample (elution peak).

[0056] Figure 20 Figure A shows the circRNA circularization efficiency detected by capillary electrophoresis (Figure A shows the untreated circRNA stock solution; Figure B shows the chromatographically purified circRNA final product).

[0057] Figure 21 Figure 1 shows the results of transfecting MSC cells with circRNA obtained by circularization (Figure A shows the positive rate of target gene expression in cells on days 3, 5, and 7 after transfection; Figure B shows the average fluorescence intensity of target gene expression in cells on days 3, 5, and 7 after transfection). Detailed Implementation

[0058] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0059] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0060] The specific steps of the LiCl precipitation method are as follows: S1. Transfer the RNA stock solution to be recovered to a 1.5 mL centrifuge tube, add an equal volume of LiCl solution and an equal volume of enzyme-free water, and refrigerate at -20℃ for more than 1 hour; after the LiCl precipitation is completed, centrifuge the centrifuge tube containing RNA at 14000 rpm for 15 min (pre-cool the centrifuge at 4℃ beforehand), and discard the supernatant after centrifugation. S2. Add an equal volume of 70% ethanol to the centrifuge tube containing the precipitate, centrifuge at 4°C and 14,000 rpm for 15 min, and discard the supernatant after centrifugation. S3. Repeat step S2, wash the RNA sample once more with 70% ethanol, place the centrifuge tube in a centrifuge and centrifuge at 14000 rpm for 3 min to thoroughly remove any residual liquid from the centrifuge tube wall, and carefully remove the supernatant. S4. Open the centrifuge tube and let it stand for 5-10 minutes to ensure that the ethanol evaporates; add an appropriate amount of enzyme-free water to resuspend the precipitate according to the upstream reaction or precipitate size, and use NanoDrop to determine the concentration.

[0061] RP-binding buffer: phosphate buffer containing 0.3M NaCl (phosphate concentration 0.05M). RP-wash buffer: phosphate buffer containing 0.1M NaCl, phosphate concentration 0.05M).

[0062] The reagents used in the following examples are listed in the table below:

[0063] The information on the type I intron self-splicing system and target gene sequences (linearized plasmid template) is shown in the table below, and the ligation diagram is as follows. Figure 1 As shown.

[0064]

[0065] Comparative Example 1: Two-step preparation of circRNA (1) In Vitro Transcription (IVT) Prepare the mRNA IVT system reaction solution according to Table 1, mix well, and place in a PCR instrument at 37℃ for 3.5h.

[0066] Table 1 mRNA in vitro transcription system

[0067] (2) Digestion of template DNA and purification of precursor RNA After the reaction was completed, 1 μL of DNase I was added and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered using an RNA purification kit to obtain the precursor circRNA.

[0068] (3) Cyclization reaction The purified precursor circRNA was heated at 70°C for 2 min and then rapidly transferred to an ice bath to cool for 2 min. Subsequently, GTP was added to the reaction solution to achieve a final concentration of 2 mM; Tris-HCl (final concentration 50 mM), MgCl2 (final concentration 10 mM), and DTT (final concentration 1 mM) were added to construct the final cyclization reaction system (pH 7.5). The cyclization reaction system was incubated at 55°C for 15 min, and the reaction solution was purified and recovered using an RNA purification kit to obtain the final circRNA (circulated circRNA).

[0069] (4) Electrophoresis detection and result analysis Precursor circRNA and final circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The results are as follows: Figure 2 As shown, the results indicate that the cyclization efficiency of circRNA in the IVT stock solution was 46.2%, and after further treatment by the cyclization reaction system, the cyclization efficiency increased to 78.5%, indicating that the components in the cyclization reaction system can improve the cyclization efficiency of circRNA.

[0070] Comparative Example 2: One-step preparation of circRNA To simplify the circRNA preparation process, the cyclization step in the two-step method was merged into the IVT step. Compared to Comparative Example 1, the mRNA in vitro transcription system increased the amount of GTP and prolonged the in vitro transcription time to simulate the cyclization reaction time in the two-step method.

[0071] (1) In vitro transcription (ITT) and RNA purification Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 1, except that the addition volume of the GTP solution (100 mM) component is 2 μL (i.e., the final GTP concentration is 10 mM). After mixing the system reaction solution, incubate it in a PCR instrument at 37℃ for 3.5 h and 16 h, respectively, to obtain circRNA products. The obtained circRNA products are purified and recovered using LiCl precipitation.

[0072] (2) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 3 As shown, the results indicate that the cyclization efficiency was only 47.4% when the IVT reaction time was 3.5 h. Further increasing the IVT reaction time to 16 h could improve the cyclization efficiency to 80.5%, indicating that the cyclization time significantly affects the cyclization efficiency.

[0073] The effect of GTP dosage on cyclization efficiency in the one-step method of Comparative Example 3 (1) In vitro transcription reaction Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 2. After mixing the system reaction solution, place it in a PCR instrument at 37°C for 16 hours.

[0074] Table 2 mRNA in vitro transcription system

[0075] (2) Digestion of template DNA and purification of circRNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain the circRNA product.

[0076] (3) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 4 As shown, the results indicate that GTP can further improve the cyclization efficiency to 82.6% when the final concentration in the IVT system is 15 mM.

[0077] Comparative Example 4: Effects of GTP and MgCl2 on cyclization efficiency in the one-step process MgCl2 was added to a one-step IVT system to investigate the effects of GTP and MgCl2 on cyclization efficiency and to explore whether cyclization efficiency can be further improved.

[0078] (1) Prepare the IVT system reaction solution according to Table 3, and add GTP and MgCl2 according to the amounts of different groups in Table 4. After mixing the IVT system reaction solution, place it in a PCR instrument at 37℃ for 3.5h.

[0079] Table 3. Amounts of each component added to the one-step IVT system

[0080] Table 4. Amounts of GTP and MgCl2 added in the one-step IVT system

[0081] Note: The in vitro transcription system contains MgCl2 at a final concentration of 20 mM. (2) Digestion of template DNA and purification of circRNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain the circRNA product.

[0082] (3) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 5 As shown, the results indicate that when the amount of GTP added was 2, 3, and 4 μL, the corresponding circRNA circulation efficiencies were 68.7%, 74.3%, and 74.0%, respectively, indicating that changing the amount of GTP did not significantly improve the circRNA circulation efficiency. When the final concentration of MgCl2 was increased to 30 mM and 40 mM, the circRNA circulation efficiencies were 70.5% and 67.5%, respectively. Compared with the control (final MgCl2 concentration of 20 mM), the yield decreased by 5.4% and 8.8%, respectively, indicating that increasing the amount of MgCl2 also did not improve the circRNA circulation efficiency, but instead led to a decrease in circRNA yield. This may be due to the additional MgCl2. 2+ It altered the salt ion balance in the transcription buffer, affecting the transcriptional activity of T7 RNA polymerase.

[0083] Comparative Example 5: Preparation of circRNA based on thermostable T7 RNA polymerase and analysis of its circularization efficiency Analysis of the cyclization reaction step in the two-step method revealed that, in addition to being related to the composition of the cyclization reaction solution, it is also correlated with the cyclization temperature. The cyclization reaction step in the two-step method is at a temperature of 55℃, which is suitable for thermostable T7 RNA polymerase. Therefore, the T7 RNA polymerase in IVT is replaced with thermostable T7 RNA polymerase.

[0084] (1) In vitro transcription reaction Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 2. Place the IVT system reaction solution in a PCR instrument and divide it into two temperature treatment groups, reacting at 50℃ and 55℃ for 3.5h.

[0085] (2) Digestion of template DNA and purification of circRNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain the circRNA product.

[0086] (3) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 6 As shown, the results indicate that the circRNA circularization efficiency obtained using the thermostable T7 RNA polymerase at 50℃ is above 92%, but the obtained RNA stock solution contains many impurities. Further increasing the reaction temperature to 55℃ prevents transcription of the target RNA. This suggests that increasing the IVT reaction temperature can improve the circRNA circularization efficiency, but the resulting circRNA contains more impurities, possibly due to excessively long IVT times leading to non-specific transcription by the T7 RNA polymerase. Furthermore, at 55℃, transcription of the target RNA fails.

[0087] Effect of in vitro transcription time on circularization efficiency in the one-step method in Comparative Example 6 In the one-step method, excessively long IVT time may lead to an increase in RNA impurities, which is not conducive to obtaining high-purity circRNA in the later stage. In order to improve circRNA purification, this invention explores whether it is possible to reduce IVT time while maintaining the same circularization efficiency.

[0088] (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 2. Place the IVT system reaction solution in the PCR instrument and divide it into two time treatment groups. React at 37℃ for 4h and 6h.

[0089] (2) Digestion of template DNA and purification of circRNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain the circRNA product.

[0090] (3) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 7As shown, the results indicate that the circularization efficiency of circRNA was 59.4%, 54.2%, 67.3%, and 70.3% when the IVT time was 4 and 6 hours, respectively, suggesting that shortening the IVT time may lead to incomplete pre-mRNA circularization.

[0091] Effect of in vitro transcription temperature on cyclization efficiency in the one-step method of Comparative Example 7 In the one-step circRNA preparation system of Comparative Example 3, further adjusting the amount of GTP and MgCl2 (Comparative Example 4) did not improve the circRNA cyclization efficiency. However, the cyclization efficiency of the thermostable T7 RNA polymerase (Comparative Example 5) shows that increasing the temperature of IVT can further improve the circRNA cyclization efficiency. Therefore, this invention explores whether increasing the reaction temperature of IVT can improve the circRNA cyclization efficiency.

[0092] (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 2, place the IVT system reaction solution in the PCR instrument, and react at 40℃ for 16h.

[0093] (2) Digestion of template DNA and purification of circRNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain the circRNA product.

[0094] (3) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 8 As shown, the results indicate that the circRNA cyclization efficiency was 92.7% and 88.4% when the reaction was carried out at 40℃ for 16 h, indicating that increasing the IVT reaction temperature can improve the circRNA cyclization efficiency.

[0095] Example 1: Effect of in vitro transcription conditions on cyclization efficiency in a one-pot method In the one-step circRNA preparation system of Comparative Example 3, reducing the IVT time leads to a decrease in the circRNA cyclization efficiency (Comparative Example 6), but increasing the IVT temperature can further improve the circRNA cyclization efficiency (Comparative Example 7). Considering that a reaction at 40℃ for 16 hours may generate a large amount of RNA impurities, it is not suitable for large-scale circRNA preparation. Therefore, this invention establishes a one-pot circRNA preparation system that takes into account the IVT reaction temperature and shortens the cyclization time while maintaining the cyclization efficiency.

[0096] (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 2, place the IVT system reaction solution in the PCR instrument, and carry out the reaction according to the reaction conditions in Table 5.

[0097] Table 5 Reaction conditions for one-pot IVT system

[0098] (2) Digestion of template DNA and purification of circRNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain the circRNA product.

[0099] (3) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 9 As shown, the results indicate that when the first-stage reaction condition is 37℃, and the second-stage incubation is at 55℃ for 1 and 2 hours, the cyclization efficiencies are 78.1% and 74.7%, respectively. When the first-stage reaction condition is 40℃, and the second-stage incubation is at 55℃ for 1 and 2 hours, the cyclization efficiencies are 90.4% and 91.1%, respectively. This shows that increasing the first-stage reaction temperature can significantly improve the cyclization efficiency, but increasing the second-stage cyclization time has no effect on improving the cyclization efficiency. Compared with the one-step method (Comparative Example 7), adding a second stage in the IVT process not only improves the circRNA cyclization efficiency but also greatly reduces the circRNA preparation time.

[0100] Example 2: Effect of T7 RNA polymerase dosage on cyclization efficiency in one-pot method The procedure was carried out based on Example 1, except that the amount of T7 RNA polymerase (50 U / µL) added was adjusted to 2, 3, 4, and 5 µL, respectively, and named Treatment Group 1 to Treatment Group 4.

[0101] (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 2. Add 2~5µL of T7 RNA polymerase (50U / µL) according to different treatment groups. After mixing the system reaction solution, place it in a PCR instrument at 40℃ for 2h and at 55℃ for 1h.

[0102] (2) Digestion of template DNA and purification of circRNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain the circRNA product.

[0103] (3) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 10 As shown, the results indicate that the circRNA cyclization efficiency of reaction systems with different amounts of T7 RNA polymerase was around 90%. The cyclization efficiencies corresponding to enzyme amounts of 2, 3, 4, and 5 µL were 89.3%, 91.3%, 90.8%, and 91.5%, respectively, indicating that increasing the amount of T7 RNA polymerase did not significantly improve the cyclization efficiency. Therefore, a small amount of T7 RNA polymerase can be used for in vitro circRNA transcription to reduce experimental costs.

[0104] Example 3: Effect of the first-stage reaction temperature on cyclization efficiency in the one-pot process Compared with Example 1, the difference is that four reaction temperature gradients were set in the first stage of the in vitro transcription reaction program, specifically, the reaction was carried out at 37, 40, 45 and 50°C for 2 hours, and the reaction was carried out at 55°C for 1 hour in the second stage.

[0105] (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 2. After mixing the system reaction solution, place it in a PCR instrument and react at 37, 40, 45 and 50℃ for 2 h, and at 55℃ for 1 h.

[0106] (2) Digestion of template DNA and purification of circRNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain the circRNA product.

[0107] (3) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 11As shown, the results indicate that adjusting the temperature of the first stage of the in vitro transcription reaction program—specifically, incubation at 37℃, 40℃, 45℃, and 50℃ for 2 hours—resulted in circRNA cyclization efficiencies of approximately 90%. The cyclization efficiencies at 37℃, 40℃, 45℃, and 50℃ were 86.3%, 90.1%, 91.5%, and 88.7%, respectively. This suggests that adjusting the reaction temperature in this stage has a limited effect on improving circRNA cyclization efficiency, and temperature changes did not cause significant differences. Considering both cyclization efficiency and experimental stability, 40℃ and 45℃ are the optimal choices.

[0108] Example 4: Effect of the first stage reaction time in the one-pot process on cyclization efficiency Compared with Example 1, the difference is that: in the first stage of the in vitro transcription reaction program, four reverse times were set, specifically 2h, 3h and 4h at 40℃ respectively, and the second stage was 1h at 55℃.

[0109] (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 2. After mixing the system reaction solution, place it in a PCR instrument and react at 40℃ for 2h, 3h, 4h, and 55℃ for 1h respectively.

[0110] (2) Digestion of template DNA and purification of circRNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain the circRNA product.

[0111] (3) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 12 As shown, the results indicate that adjusting the reaction time of the first stage of the in vitro transcription reaction program, specifically incubation at 40℃ for 2 h, 3 h, and 4 h, resulted in circRNA circulation efficiencies of 91.6%, 90.4%, and 90.5%, respectively. This demonstrates that extending the reaction time in this stage did not significantly improve circulation efficiency, and the efficiency difference was not statistically significant. Considering both circulation efficiency and reaction time, 2 h was selected as the optimal condition, which can significantly shorten the reaction time while ensuring efficient circulation.

[0112] Example 5: Effect of the reaction temperature in the second stage of the one-pot process on cyclization efficiency Compared with Example 1, the difference is that the in vitro transcription reaction procedure is as follows: the first stage is a 2-hour reaction at 40°C, and the second stage is set with four reaction temperature gradients, specifically incubation at 50°C, 55°C, 60°C and 65°C for 1 hour respectively.

[0113] (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 2. After mixing the system reaction solution, place it in a PCR instrument at 40℃ for 2h, and then react at 50℃, 55℃, 60℃ and 65℃ for 1h respectively.

[0114] (2) Digestion of template DNA and purification of circRNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain the circRNA product.

[0115] (3) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 13 As shown, the results indicate that adjusting the reaction temperature of the second stage of the in vitro transcription reaction program—specifically, 50℃, 55℃, 60℃, and 65℃ for 1 h—resulted in significantly different circRNA cyclization efficiencies. The cyclization efficiencies at 50℃, 55℃, 60℃, and 65℃ were 91.6%, 90.9%, 84.2%, and 83.7%, respectively. This demonstrates that excessively high reaction temperatures in the second stage significantly reduce cyclization efficiency; therefore, the temperature for this step should not be set too high. In summary, 50℃ and 55℃ are the optimal reaction conditions, maintaining both high cyclization efficiency and ensuring stable reaction progress.

[0116] Example 6: Effect of the second-stage reaction time in the one-pot process on cyclization efficiency Compared with Example 1, the difference is that three reverse times are set in the second stage of the in vitro transcription reaction program. Specifically, the first stage of the in vitro transcription reaction program is 40°C for 2 hours, and the second stage is 55°C for 0.5 hours, 1 hour, and 1.5 hours respectively.

[0117] (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 2. After mixing the system reaction solution, place it in a PCR instrument and react at 40℃ for 2h, and at 55℃ for 0.5h, 1h and 1.5h respectively. (2) Digestion of template DNA and purification of circRNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain the circRNA product.

[0118] (3) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 14 As shown, the results indicate that adjusting the reaction time of the second stage of the in vitro transcription reaction program, specifically reacting at 55℃ for 0.5 h, 1 h, and 1.5 h, did not significantly affect the circRNA cyclization efficiency. The cyclization efficiencies for 0.5 h, 1 h, and 1.5 h were 91.4%, 91.8%, and 90.8%, respectively. This suggests that the reaction time in this stage, within the range of 0.5 to 1.5 h, has little impact on cyclization efficiency. Considering both reaction efficiency and time cost, 0.5 h or 1 h is the optimal condition, effectively shortening the reaction cycle while ensuring high cyclization efficiency.

[0119] Example 7: Effect of GTP Dosage on Cycloning Efficiency in One-Pot Process Compared with Example 1, the difference is that the amount of GTP solution (100 mM) added to the in vitro transcription (IVT) system reaction solution was adjusted so that the final GTP concentrations were 12.5 mM, 15 mM, 17.5 mM and 20 mM, respectively.

[0120] (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 2, the difference being: adjust the amount of GTP solution (100 mM) added so that the final GTP concentrations are 12.5 mM, 15 mM, 17.5 mM, and 20 mM respectively. After mixing the system reaction solution, place it in a PCR instrument at 40℃ for 2 h and at 55℃ for 1 h.

[0121] (2) Digestion of template DNA and purification of circRNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template. After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain the circRNA product.

[0122] (3) Electrophoresis detection and result analysis The purified and recovered circRNA samples were subjected to 3% agarose gel electrophoresis, and the bands were analyzed using ImageJ software. The analysis results are as follows: Figure 15As shown, the results indicate that there are significant differences in the circRNA cyclization efficiency prepared with different amounts of GTP. The cyclization efficiencies corresponding to the final GTP concentrations of 12.5 mM, 15 mM, 17.5 mM, and 20 mM are 88.8%, 91.8%, 62.9%, and 20.4%, respectively. This suggests that a slight increase in the amount of GTP can promote circRNA cyclization, but it should not be excessive. For example, the cyclization efficiency decreases rapidly when the final concentrations are 17.5 mM and 20 mM.

[0123] Example 8: Effect of one-pot R-buffer method on the efficiency of RNase R digestion of linear RNA Both the two-step and current one-step methods for circRNA preparation require changing the reaction tube and purifying the circRNA stock solution after DNase I treatment, followed by RNase R treatment. This process is cumbersome and prone to circRNA loss during repeated operations. This invention develops a novel reaction buffer (Rbuffer) that eliminates the need for circRNA stock solution purification. The Rbuffer can be directly added to the DNase I-treated reaction system, followed by RNase R for efficient removal of linear RNA impurities. This method simplifies the procedure, significantly improves experimental efficiency, reduces circRNA loss, and offers greater practicality and stability. The specific steps are as follows: (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 6. After mixing the system reaction solution, place it in a PCR instrument at 40℃ for 2 hours and at 55℃ for 1 hour.

[0124] Table 6 mRNA in vitro transcription system

[0125] (2) Digestion of template DNA After the in vitro transcription reaction was completed, 1 μL of DNase I was added to the reaction system and incubated at 37°C for 1 h to digest the linearized plasmid DNA template.

[0126] (3) Prepare RNase R reaction buffers (R Buffer) for different treatment groups according to Table 7, using commercial RNase R Buffer as a control. After mixing each reaction system, add 10 U RNase R and incubate at 37℃ for 15 minutes to degrade linear RNA impurities. Wherein: The control group (number 1) used linear RNA samples purified by LiCl, diluted with enzyme-free water using RNase R Buffer; Experimental groups (numbered 2-8) directly used the original solution after in vitro transcription (IVT) in step (2), without purification, and directly added the corresponding RNase R reaction buffer to the reaction system for subsequent processing.

[0127] Table 7 RNase R reaction buffer for different treatment groups

[0128] (4) After the reaction in step (3) is completed, the sample is directly subjected to 3% agarose gel electrophoresis for detection, and the bands are analyzed using ImageJ software. The detection and analysis results are as follows: Figure 16 As shown in Table 8, the results indicate that, without purification of the IVT stock solution, the use of a mixed solution containing 2M MgCl2 and KCl, a mixed solution containing 2M MgCl2 and LiCl, or the addition of 2M MgCl2 solution or 2M LiCl solution as RNase R reaction buffer can effectively support RNase R activity, and its degradation efficiency for linear RNA is better than that of commercial RNase R buffer.

[0129] Table 8. ImageJ software analysis of RNase R activity in different reaction buffers.

[0130] Example 9: Effect of Rbuffer dosage on the efficiency of RNase R digestion of linear RNA in one-pot digestion This example uses LiCl solution to illustrate the effect of Rbuffer dosage on the efficiency of RNase R digestion of linear RNA.

[0131] (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 6. After mixing the system reaction solution, place it in a PCR instrument at 40℃ for 2h and at 55℃ for 1h. (2) After the reaction was completed, 1 μL of DNase I was added and incubated at 37°C for 1 h to digest the plasmid template. (3) Prepare LiCl solutions with concentrations of 1, 2, and 3 M, take 2 μL and add it to the IVT reaction solution after the reaction in step (2). After mixing, add 10 U of RNase R and incubate at 37°C for 15 min to digest linear RNA. (4) After the reaction in step (3) is completed, the sample is directly subjected to 3% agarose gel electrophoresis for detection, and the bands are analyzed using ImageJ software. The detection and analysis results are as follows: Figure 17 As shown, the results indicate that adding 1M, 2M, and 3M LiCl solutions to the IVT reaction solution can maintain the activity of RNase R and eliminate linear RNA impurities.

[0132] Example 10: Effect of oligo dT purification system on circRNA purification efficiency To verify the feasibility of process scale-up, the amount of each component in the mRNA in vitro transcription system was increased by 200 times to prepare mg-level circRNA. CircRNA was prepared under suboptimal IVT conditions to verify the efficiency of RNaseR in removing linear RNA impurities and the overall purification process's effect on impurity removal.

[0133] (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 9. After mixing the system reaction solution, place it in a metal bath and react at 37°C for 1 hour and 55°C for 1 hour.

[0134] Table 9 mRNA in vitro transcription system

[0135] (2) Digestion of template DNA After the in vitro transcription reaction was completed, 200 μL of DNase I was added to the reaction system and incubated at 37°C for 1 hour to digest the linearized plasmid DNA template.

[0136] (3) Digestion of linear RNA Prepare a 2M LiCl solution, take 200 μL and add it to the IVT reaction solution after the reaction in step (2). After mixing, add 2000U of RNase R and incubate at 37℃ for 15 min to digest linear RNA.

[0137] (4) The circRNA stock solution treated with RNaseR was filtered through a 0.22 μm filter membrane. The filtrate was transferred to a new 15 mL centrifuge tube. Then, 1 / 3 volume of RP-binding buffer was added to the filtrate, mixed well, and placed on ice for later use.

[0138] (5) Set the circular mRNA purification method according to the parameters in Table 10. The purification was carried out by loading the sample using a system pump. The sample collection at each stage was carried out in signal collection mode with the detection parameter being U260. The column pressure alarm was set to 0.5 MPa throughout the purification process.

[0139] Table 10. Main parameters for circular mRNA purification

[0140] (6) Connect RP-binding buffer, RP-rinsing buffer, enzyme-free water, 0.1M NaOH and 20% ethanol to the A1, A2, A3, A4 and B1 pathways respectively, and use manual air removal method to remove air from each pathway and replace the buffer solution of each pathway.

[0141] (7) Connect the chromatographic column to the chromatographic purification system and call the manual operation command to use 100% A1 to balance the dT20 chromatographic column for 5-10 column volumes.

[0142] (8) Connect the A5 channel to the sample pretreatment solution, call the circular mRNA purification method file, and start the purification operation.

[0143] (9) After mixing the collected sample peaks by inverting them, take 2 μL of each sample for RNA concentration determination (Qubit4), and store the remaining samples in a -20℃ refrigerator.

[0144] (10) The obtained circRNA samples were detected by 3% agarose gel electrophoresis and capillary electrophoresis was used for analysis to accurately quantify the circumduction efficiency of circRNA.

[0145] Test results as follows Figure 18 , Figure 19 and Figure 20 As shown, the results indicate that under suboptimal IVT conditions, the circRNA cyclization efficiency was only 76.49%. After RNaseR treatment, pre-mRNA impurities could be completely eliminated, but some small intron fragments generated during cyclization could not be removed. The obtained circRNA stock solution was affinity purified. As can be seen from the chromatogram, there was no crossflow during the entire purification process, the elution peak was single, and the obtained circRNA final product had a single band without linear RNA impurities. CE detection of the final product showed that the circRNA cyclization efficiency was 98.61% and the integrity was 97.42%.

[0146] Example 11: Analysis of the results of transfecting MSC cells with circRNA obtained by circularization The purpose of this embodiment is to characterize the cell expression effect of the prepared circRNA.

[0147] (1) Prepare the mRNA in vitro transcription (IVT) system reaction solution according to Table 6. After mixing the system reaction solution, place it in a PCR instrument at 40℃ for 2h and at 55℃ for 1h. (2) After the reaction was completed, 1 μL of DNase I was added and incubated at 37°C for 1 h to digest the linear plasmid template; (3) Take 2 μL of 2M LiCl solution and add it to the IVT reaction solution after the reaction in step (2). After mixing, add 10U of RNaseR and incubate at 37℃ for 15 min to digest linear RNA. (4) After the reaction was completed, the product was purified and recovered by LiCl precipitation to obtain circRNA product.

[0148] The cells used in this embodiment are human bone marrow mesenchymal stem cells (MSCs), and the culture media used are all serum-free culture media.

[0149] I. Experimental Methods and Procedures 1. Cell resuscitation (1) Remove the frozen cells from the liquid nitrogen and place them at -80℃ for 5 min to evaporate the residual liquid nitrogen; at the same time, warm the culture medium to room temperature (≥30 min) and preheat the water bath to 39.0℃.

[0150] (2) Quickly place the cryovial into a water bath and gently shake for about 60 seconds (≤2 min) to complete the thawing.

[0151] (3) Transfer the cryopreservation tube to the biosafety cabinet, disinfect it with 75% alcohol, open the lid, and transfer the cell suspension into a 15 mL centrifuge tube containing 4 mL of rewarmed culture medium.

[0152] (4) Centrifuge at 400×g for 5 min, discard the supernatant, add 1 mL of culture medium to resuspend the cells, and take 20 μL of the cell suspension for cell counting.

[0153] (5) Centrifuge at 400×g for 5 min, discard the supernatant, add 10 mL of culture medium, and gently pipette until the cells are completely dispersed into single cells (no more than 10 pipette cycles). (6) Cell seeding, usually at 1×10 4 ~2×10 4 cells / cm 2 Inoculate into T150 culture flasks for culture, using 20 mL of culture medium per T150 flask; (7) Shake the culture flask in a cross pattern of "front-back-left-right" to disperse the cells evenly, and culture at 37°C with 5% CO2.

[0154] 2. Cell electroporation (1) Preparation before the experiment: Take out the culture medium in advance to warm it up, turn off the biosafety cabinet after irradiating it with ultraviolet light for at least 30 minutes, and wipe the surface of the biosafety cabinet with alcohol.

[0155] (2) Perform cell digestion on the cultured cells; (3) After digestion, the cell suspension was centrifuged at 400 ×g for 5 min, the supernatant was removed, 1 mL of culture medium was added to resuspend the cells, and 20 μL of cell suspension was taken for cell counting. (4) Centrifuge the cell suspension at 400 ×g for 5 min, remove the supernatant, add 10 mL of physiological saline to resuspend the cells, centrifuge at 400 ×g for 4 min, and discard the supernatant.

[0156] (5) Calculate the required volume of EL electroporation buffer based on the cell count results, and resuspend the cell pellet. (6) According to the experimental design, take an appropriate amount of cell suspension resuspended in EL electroporation buffer, and administer 2 μg circRNA / 1*10 cells. 6 Cell preparation, mix thoroughly using a pipette; (7) Transfer the cell suspension required for electroporation to the electrode cup and set the electroporation parameters according to Table 11.

[0157] Table 11 Electrical Transfer Parameters

[0158] (8) After electroporation, the cells need to be counted, and the cell viability and total number of cells should be recorded. Then, the cells are counted again at a rate of 2 × 10⁻⁶. 4 cells / cm 2 Inoculate into T25 culture flasks, adding 4 mL of culture medium per flask. Then transfer the culture flasks to a CO2 incubator and incubate at 37°C with 5% CO2.

[0159] 3. Cell flow cytometry detection (1) Preparation before the experiment: Take out the culture medium in advance to warm it up, turn off the biosafety cabinet after irradiating it with ultraviolet light for at least 30 minutes, and wipe the surface of the biosafety cabinet with alcohol. (2) Perform cell digestion on the cultured cells; (3) After digestion, the cell suspension was centrifuged at 400×g for 5 min, the supernatant was removed, and the cells were resuspended in 1 mL of PBS buffer. (4) Centrifuge at 400×g for 4 min, remove the supernatant, add 200 μL PBS, and aliquot each group of cells and blank unpaired cells into two flow cytometry tubes, labeled as: sample name-IC and sample name-CXCR5 respectively. Add antibody according to Table 12. Table 12 Reference Table for Antibody Addition Amount

[0160] (5) Mix thoroughly and incubate at room temperature in the dark for 30 min; (6) Add 1 mL of PBS and mix well. Centrifuge at 400×g for 4 min and remove the supernatant.

[0161] (7) Add 200 μL PBS to resuspend the cells for flow cytometry detection.

[0162] 4. Data Processing (1) Flow cytometry data processing: The flow cytometry results were imported into CytExpert / FlowJo V10 software for analysis. Live cells were gated in the scatter plot and labeled as P1 group. Single cells of P1 group were gated in the scatter plot with SSC-A on the x-axis and SSC-H on the y-axis and labeled as P2 group. P2 group cells of the control group were marked with lines in the histogram with APC-A on the x-axis and COUNT on the y-axis to mark negative and positive cells. Positive areas were marked as P3. The positive rate and mean fluorescence value (MFI) of P3 group cells in the antibody group were calculated.

[0163] (2) Statistical data processing: The positive rate and MFI value calculated by CytExpert / FlowJo V10 were input into Prism software. The differences in positive rate and average fluorescence value among different groups after electroporation and culture for the same period of time were analyzed by univariate variable analysis.

[0164] Test results as follows Figure 21 As shown, the results indicate that the protein expression positivity rate of the prepared circRNA was still 80% on day 7 after entering the cells, and the MFI was 200,000, indicating that the circRNA has strong and sustained expression in the cells.

[0165] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing circRNA, characterized in that, Includes the following steps: (1) The in vitro transcription reaction system of circRNA was subjected to a two-stage reaction: The circRNA in vitro transcription reaction system was subjected to the first stage reaction at 37–50°C for 1–4 h; then the second stage reaction was carried out at 50–65°C for 0.5–1.5 h. (2) Enzyme treatment: The reaction products were sequentially treated with DNA enzyme and RNA enzyme; (3) Purify to obtain circRNA; The two-stage reaction, enzyme treatment, and purification are all completed in the same reaction vessel.

2. The method according to claim 1, characterized in that, The temperature of the first stage of in vitro transcription reaction is 40~45℃, and the reaction time is 2~4 hours.

3. The method according to claim 1, characterized in that, The temperature for the second stage of in vitro transcription reaction is 50-55℃, and the reaction time is 0.5-1 hour.

4. The method according to claim 1, characterized in that, The RNase treatment is performed by adding RNase buffer and RNase to the reaction system; the RNase buffer is any one of the following: a mixed solution of MgCl2 and KCl, a mixed solution of MgCl2 and LiCl, a MgCl2 solution, and a LiCl solution.

5. The method according to claim 4, characterized in that, The volume ratio of the RNase buffer to the reaction system is (0.5~1.5):

10.

6. The method according to claim 1, characterized in that, The RNase treatment conditions are: 35~39℃ for 20~40 minutes.

7. The method according to claim 1, characterized in that, The DNase treatment is as follows: add DNase to the reaction system and incubate at 35~39℃ for 20~40 minutes.

8. The method according to claim 1, characterized in that, The in vitro transcription reaction system comprises the following components: T7 RNA polymerase transcription buffer, UTP, ATP, CTP, GTP, T7 RNA polymerase, inorganic pyrophosphatase, linear plasmid template, and water.

9. The method according to claim 8, characterized in that, The final concentration of GTP in the in vitro transcription reaction system is 12.5~17.5mM.

10. The method according to claim 8, characterized in that, The final concentration of T7 RNA polymerase in the in vitro transcription reaction system is 5~12.5 U / μL.

Images