Preparation method of circular nucleic acid

Abstract

The invention discloses a preparation method of circular nucleic acid. A transcription vector construction module, a cyclization sequence design module, an in-vitro transcription module, a cyclization module and a purification module are included. The transcription vector construction module constructs a transcription vector containing a promoter, a target gene and a cyclization sequence, and the promoter is located at the upstream of the target gene; a cyclization sequence designed by the cyclization sequence design module is connected with a target gene in the transcription vector; the in-vitro transcription module takes a transcription vector as a template to generate linear nucleic acid molecules; the cyclization module cyclizes the linear nucleic acid molecules to form annular nucleic acid; the purification module is used for purifying the cyclized product; the transcription vector construction module is responsible for constructing a transcription vector containing a promoter, a target gene and a cyclization sequence, and the promoter is located at the upstream of the target gene and drives a transcription process. The cyclization efficiency is greatly improved, for example, the cyclization efficiency of an intron self-shearing method is lower than 50%, and the cyclization efficiency of a T4 ligase method is 60%-70%; exogenous residues are obviously reduced, and the process steps are reduced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing circular nucleic acids. Background Technology

[0002] The preparation of circular nucleic acids involves multiple interdisciplinary technologies. In gene therapy, circular nucleic acids can serve as ideal gene carriers for delivering therapeutic genes; in vaccine development, they can encode antigens and stimulate the body's immune response, making them a promising key technology for next-generation vaccines. Furthermore, they also have potential applications in disease diagnosis and gene regulation.

[0003] Circular nucleic acids (circular RNA and circular single-stranded DNA) have shown broad application prospects in biomedical fields such as disease diagnosis and treatment, and gene regulation due to their unique closed-circular structure and excellent biological stability. For example, in disease treatment, circular RNA can be used in the development of lung cancer vaccines, bringing new hope for the treatment of osimertinib-resistant lung cancer; circular nucleic acid technology also has potential application value in the development of COVID-19 vaccines. In the field of gene regulation, circular nucleic acids can perform specific functions such as miRNA sponges, protein sponges, and gene silencing.

[0004] However, existing circular nucleic acid preparation technologies have many shortcomings and are difficult to meet the growing application demands. The following is a comparison of the efficiency and limitations of different existing methods: 1. The cyclization efficiency of the intron self-cleavage method is low, sometimes below 50%, with a certain amount of exogenous sequence residue. The process is relatively complex, requiring specific intron sequences and conditions. Low cyclization efficiency limits yield, and exogenous residue may affect subsequent applications. 2. The cyclization efficiency of the T4 ligase method is generally 60%-70%. It easily introduces exogenous sequences, resulting in high residue levels. The process involves many steps, requiring strict control of reaction conditions. Furthermore, the cyclization efficiency needs improvement, the exogenous residue problem is prominent, and the complex process is not conducive to large-scale production.

[0005] The shortcomings of these existing technologies mean that the quality and yield of the prepared circular nucleic acids are insufficient to meet the needs of practical applications. Low cyclization efficiency limits the yield of circular nucleic acids, making large-scale production difficult; exogenous sequence residues may affect the bioactivity and safety of circular nucleic acids; and complex processes increase preparation costs and time, reducing production efficiency. Therefore, developing an efficient, low-residue, and simple method for preparing circular nucleic acids is of significant practical importance. Summary of the Invention

[0006] In view of the problems raised in the background art above, the purpose of this invention is to provide a method for preparing circular nucleic acids. This invention focuses on developing an efficient and reliable in vitro preparation method for circular nucleic acids to meet the demand for large quantities of high-quality circular nucleic acids in biomedical research and applications. Through the preparation method of this invention, high-quality circular nucleic acid materials can be provided for these fields, promoting the development of related technologies.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing circular nucleic acids includes a transcription vector construction module, a circular sequence design module, an in vitro transcription module, a circularization module, and a purification module;

[0009] The transcription vector construction module constructs a transcription vector containing a promoter, a target gene, and a circularized sequence, with the promoter located upstream of the target gene; the circularized sequence design module designs a circularized sequence that is linked to the target gene in the transcription vector; the in vitro transcription module uses the transcription vector as a template to generate linear nucleic acid molecules; the circularization module circularizes the linear nucleic acid molecules to form circular nucleic acids; and the purification module purifies the circularized product.

[0010] The transcription vector construction module is responsible for constructing a transcription vector containing a promoter, a target gene, and a circular sequence. The promoter is located upstream of the target gene and drives the transcription process.

[0011] The circular sequence design module designs circular sequences to guide nucleic acid molecules to form closed circular structures, and the circular sequences are closely linked to the target gene in the transcription vector;

[0012] The in vitro transcription module is directly connected to the transcription vector construction module;

[0013] The cyclization module uses a specific enzyme or self-cleavage mechanism to cyclize linear nucleic acid molecules into circular nucleic acids. The cyclization module receives linear nucleic acids generated by the in vitro transcription module and works in synergy with the cyclization sequence design module. The cyclization sequence guides the cyclization reaction.

[0014] The purification module removes impurities, unreacted raw materials, and exogenous sequences from the reaction system to obtain high-purity circular nucleic acid. The purification module is connected to the circularization module.

[0015] Further specifying, in the transcription vector construction module, the promoter is the T7 promoter, which specifically binds to RNA polymerase to drive transcription initiation, and the promoter and the target gene are connected by complementary base pairing. In the transcription vector construction module, the vector is pUC19, and the vector and the sequence containing type II introns and the target gene are ligated by ligase at 37°C for 2 hours.

[0016] Further specifying, in the circularization sequence design module, the circularization sequence is selected from any one of SEQ ID NO.2-9, and the circularization sequence guides the nucleic acid molecule to form a closed loop through base complementary pairing and spatial conformation.

[0017] Further specifying, in the in vitro transcription module, T7 thermostable RNA polymerase is used for transcription, which is performed in vitro at 37-52℃. The in vitro transcription module and the transcription vector construction module are connected through the transfer of the transcription vector.

[0018] Further specifying, the circularization module employs either a type II intron self-cleavage mechanism or a T4 RNA ligase method for circularization; if the type II intron self-cleavage mechanism is used, different domains of the type II intron cooperate to achieve self-cleavage and circularization; if the T4 RNA ligase method is used, the T4 RNA ligase catalyzes the formation of a phosphodiester bond between the 5' phosphate group and the 3' hydroxyl group of the linear nucleic acid molecule to achieve circularization, and the circularization module receives the linear nucleic acid generated by the in vitro transcription module and works synergistically with the circularization sequence design module;

[0019] In the circularization module, if the T4 RNA ligase method is used, the reaction is carried out overnight at 16°C. The T4 RNA ligase and the linear nucleic acid molecule are circularized by specifically binding to and recognizing the sequences at both ends.

[0020] Further specifying, the purification module uses column chromatography or a continuous flow chromatography system for purification to remove impurities, unreacted raw materials and exogenous sequences from the reaction system. The purification module and the cyclization module are connected through the transfer of cyclization products.

[0021] In the purification module, when column chromatography is used for purification, the circular nucleic acid and impurities are separated and purified according to their different physicochemical properties.

[0022] Further specifying, the prepared circular nucleic acid is either circular RNA or circular DNA. When preparing circular RNA, it can be used for vaccine antigen expression or CAR-T encoding; when preparing circular DNA, it can be used for miRNA sponges or diagnostic probes.

[0023] Further, in large-scale preparation, a 10L reaction system is used, and the vector is prepared in batches by culturing E. coli containing the transcription vector in a fermenter and extracting the vector DNA by alkaline lysis. Automated circularization is carried out using a bioreactor and the reaction conditions are controlled by an automated control system. Continuous purification is carried out using a continuous flow chromatography system, and the purity of the prepared circular nucleic acid is ≥95% and the endotoxin is ≤0.1EU / mL.

[0024] To further specify, when preparing circular RNA for lung cancer vaccines, the vector construction involves inserting a sequence containing type II introns and the lung cancer antigen gene into the pUC19 vector, in vitro transcription is incubated at 37°C for 3 hours, circularization is performed at 37°C for 2 hours, and purification is carried out using column chromatography.

[0025] Further specifying that, when preparing circular DNA for liver cancer treatment, the linear DNA is designed to contain the SEQ ID NO.1 sequence that can bind complementary to oncogenic miRNA, the enzymatic circularization is performed using T4 RNA ligase overnight at 16°C, and RNase R digestion is performed at 37°C for 1 hour.

[0026] The beneficial effects of this invention are:

[0027] The cyclization efficiency of this invention is significantly improved compared to existing technologies, such as the intron self-cleavage method (cyclization efficiency below 50%) and the T4 ligase method (cyclization efficiency 60%-70%). Exogenous residues are significantly reduced, far below the high levels easily introduced by existing technologies. The reduced number of process steps greatly simplifies the preparation process, and these advantages positively promote downstream applications. Furthermore, in vaccine development, the improved stability of circular nucleic acids makes vaccines more reliable; in gene therapy, reduced costs improve the accessibility of gene therapy. For example, in lung cancer vaccines and CAR-T cell therapy, the circular nucleic acids prepared by this invention better meet application requirements. Attached Figure Description

[0028] The present invention can be further illustrated by the non-limiting embodiments given in the accompanying drawings;

[0029] Figure 1 This is a system block diagram of an embodiment of a method for preparing circular nucleic acids according to the present invention;

[0030] Figure 2 This is a flowchart illustrating the steps of an embodiment of a method for preparing circular nucleic acids according to the present invention.

[0031] Figure 3 This is a flowchart illustrating the steps of an embodiment of a method for preparing circular nucleic acids according to the present invention.

[0032] The symbols of the main components are explained as follows: transcription vector construction module 101, circular sequence design module 102, in vitro transcription module 105, circularization module 103, and purification module 104. Detailed Implementation

[0033] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments. The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0034] It should be noted that all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indication will also change accordingly.

[0035] Furthermore, the use of terms such as "first" and "second" in this invention is for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. If the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0036] like Figure 1 As shown, a method for preparing circular nucleic acids according to the present invention includes a transcription vector construction module 101, a circular sequence design module 102, an in vitro transcription module 105, a circularization module 103, and a purification module 104.

[0037] The transcription vector construction module 101 constructs a transcription vector containing a promoter, a target gene, and a circularized sequence, with the promoter located upstream of the target gene; the circularized sequence design module 102 designs a circularized sequence that is linked to the target gene in the transcription vector; the in vitro transcription module 105 generates a linear nucleic acid molecule using the transcription vector as a template; the circularization module 103 circularizes the linear nucleic acid molecule to form a circular nucleic acid; and the purification module 104 purifies the circularized product.

[0038] The transcription vector construction module 101 is responsible for constructing a transcription vector containing a promoter, a target gene, and a circular sequence. The promoter is located upstream of the target gene and drives the transcription process.

[0039] The circular sequence design module 102 designs a circular sequence to guide nucleic acid molecules to form a closed circular structure, and the circular sequence is closely linked to the target gene in the transcription vector;

[0040] The in vitro transcription module 105 is directly connected to the transcription vector construction module 101;

[0041] The cyclization module 103 uses a specific enzyme or self-cleavage mechanism to cyclize linear nucleic acid molecules into circular nucleic acids. The cyclization module 103 receives linear nucleic acids generated by the in vitro transcription module and works in synergy with the cyclization sequence design module 102. The cyclization sequence guides the cyclization reaction.

[0042] The purification module 104 removes impurities, unreacted raw materials, and exogenous sequences from the reaction system to obtain high-purity circular nucleic acid. The purification module 104 is connected to the cyclization module 103.

[0043] In the practical application of this embodiment, in the transcription vector construction module 101, the promoter is the T7 promoter, which specifically binds to RNA polymerase to drive transcription initiation, and the promoter and the target gene are connected by complementary base pairing. In the transcription vector construction module 101, the vector is pUC19, and the vector and the sequence containing type II introns and the target gene are ligated by ligase at 37°C for 2 hours.

[0044] In the practical application of this embodiment, in the circularization sequence design module 102, the circularization sequence is selected from any one of SEQ ID NO.2-9, and the circularization sequence guides the nucleic acid molecule to form a closed loop through base complementary pairing and spatial conformation.

[0045] In the practical application of this embodiment, the in vitro transcription module 105 uses T7 thermostable RNA polymerase for transcription. This enzyme performs in vitro transcription at 37-52°C. The in vitro transcription module 105 and the transcription vector construction module 101 are connected through the transfer of the transcription vector.

[0046] In the practical application of this embodiment, the circularization module 103 uses either a type II intron self-cleaving mechanism or a T4 RNA ligase method for circularization. If the type II intron self-cleaving mechanism is used, the different domains of the type II intron cooperate to achieve self-cleavage and circularization. If the T4 RNA ligase method is used, the T4 RNA ligase catalyzes the formation of a phosphodiester bond between the 5' phosphate group and the 3' hydroxyl group of the linear nucleic acid molecule to achieve circularization. The circularization module 103 receives the linear nucleic acid generated by the in vitro transcription module 105 and works synergistically with the circularization sequence design module 102.

[0047] In the circularization module 103, if the T4 RNA ligase method is used, the reaction is carried out overnight at 16°C. The T4 RNA ligase and the linear nucleic acid molecule recognize the sequences at both ends through specific binding and circularization.

[0048] In the practical application of this embodiment, the purification module 104 uses column chromatography or a continuous flow chromatography system to purify the reaction system, removing impurities, unreacted raw materials and exogenous sequences. The purification module and the cyclization module are connected through the transfer of cyclization products.

[0049] In the purification module 104, when column chromatography is used for purification, the circular nucleic acid and impurities are separated and purified according to their different physicochemical properties.

[0050] In the practical application of this embodiment, the prepared circular nucleic acid is circular RNA or circular DNA. When circular RNA is prepared, it can be used for vaccine antigen expression or CAR-T encoding; when circular DNA is prepared, it can be used for miRNA sponges or diagnostic probes.

[0051] In the practical application of this embodiment, a 10L reaction system is used for large-scale preparation. The vector is prepared in batches by culturing E. coli containing the transcription vector in a fermenter and extracting the vector DNA by alkaline lysis. Automated circularization is carried out using a bioreactor and the reaction conditions are controlled by an automated control system. Continuous purification is carried out using a continuous flow chromatography system, and the purity of the prepared circular nucleic acid is ≥95% and the endotoxin is ≤0.1EU / mL.

[0052] In the practical application of this embodiment, when preparing circular RNA for lung cancer vaccine, the vector construction involves inserting a sequence containing type II introns and lung cancer antigen genes into the pUC19 vector, in vitro transcription is incubated at 37°C for 3 hours, circularization is performed at 37°C for 2 hours, and purification is carried out using column chromatography.

[0053] In the practical application of this embodiment, when preparing circular DNA for liver cancer treatment, the linear DNA is designed to contain the SEQ ID NO.1 sequence that can bind complementary to the oncogenic miRNA. Enzymatic circularization is performed using T4 RNA ligase overnight at 16°C, and RNase R digestion is carried out at 37°C for 1 hour.

[0054] The working principle of this invention is as follows:

[0055] The working principle of this invention for preparing cyclic nucleic acids is based on molecular mechanisms and the functional characteristics of carrier elements in biochemistry. Regarding the cyclization reaction, if a type II intron self-cleavage mechanism is employed, its molecular mechanism relies on the synergistic effect of its domains. Type II introns possess specific secondary and tertiary structures. Under suitable conditions, different domains cooperate to allow the intron to self-cleave from the linear nucleic acid molecule, while the nucleic acid fragments at both ends connect to form a closed loop. This process involves interactions such as base pairing and hydrogen bond formation within the nucleic acid molecule, prompting conformational changes and ultimately achieving cyclization.

[0056] If enzymatic ligation is used, such as with T4 RNA ligase, the principle is to achieve cyclization by catalyzing the formation of phosphodiester bonds. The enzyme binds to the linear nucleic acid molecule, recognizes specific sequences at both ends, and links the 5' phosphate group to the 3' hydroxyl group, forming a stable phosphodiester bond, thereby cyclizing the linear nucleic acid molecule.

[0057] In terms of vector elements, the promoter, as a key regulatory element of gene expression, can specifically bind to RNA polymerase, driving the initiation of transcription. It provides the initiation signal for transcription, enabling RNA polymerase to accurately recognize and bind to the transcription start site, thereby initiating the synthesis of linear nucleic acid molecules. The circularization sequence plays a guiding role in the cyclization process. It has a specific base sequence and secondary structure, which can interact with specific regions of the nucleic acid molecule, guiding the nucleic acid molecule to form a closed circular structure, ensuring the efficient execution of the cyclization reaction.

[0058] Example 1: Preparation of circular RNA based on type II introns

[0059] In the context of osimertinib-resistant lung cancer treatment, a circular RNA for lung cancer vaccine development was prepared. First, a vector was constructed by inserting a sequence containing a type II intron and a lung cancer antigen gene into a suitable vector, pUC19, and the ligation reaction was carried out at 37°C for 2 hours. Next, in vitro transcription was performed using the constructed vector as a template, adding enzymes and substrates required for transcription, such as T7 RNA polymerase, and incubating at 37°C for 3 hours to generate linear RNA molecules. Then, a circularization step was performed, placing the transcription product in a specific buffer and reacting at 37°C for 2 hours, utilizing the self-cleaving mechanism of the type II intron to circularize the linear RNA. Finally, purification was performed, using column chromatography to remove impurities, yielding high-purity circular RNA. This circular RNA can serve as an antigen expression vector for a lung cancer vaccine, providing a new strategy for the treatment of osimertinib-resistant lung cancer.

[0060] Example 2: Preparation of circular DNA based on T4 RNA ligase

[0061] To address the need for inhibiting oncogenic miRNAs in liver cancer treatment, circular DNA was prepared. First, linear DNA containing a sequence (SEQ ID NO. 1) complementary to the oncogenic miRNA was designed. Then, enzymatic circularization was performed by mixing the linear DNA with T4 RNA ligase in a buffer solution and reacting overnight at 16°C to form circular DNA. Next, RNase R digestion was performed by adding RNase R enzyme and digesting at 37°C for 1 hour to remove uncirculated linear RNA. These steps yielded circular DNA for liver cancer treatment. This circular DNA can act as a miRNA sponge, specifically binding to oncogenic miRNAs and inhibiting their activity, thereby achieving the therapeutic goal of liver cancer.

[0062] Example 3: Preparation of long circular RNA with no exogenous residue (CAR-T encoding)

[0063] To achieve in situ generation of CAR-T cells, the preparation of long-chain (2000 nt) circular RNA was optimized. First, the circularization sequence was redesigned to better facilitate the circularization of long-chain RNA and reduce foreign sequence residue. Then, transcription was performed using a thermostable T7 polymerase at 65°C. Compared to traditional T7 polymerase, the thermostable T7 polymerase is more efficient at transcribing long-chain RNA. Compared to traditional methods, the circularization efficiency increased from 60% to over 90%, and foreign residue decreased from 15% to below 5%. The prepared long-chain circular RNA can be used to encode proteins required for CAR-T cells, enabling in situ generation of CAR-T cells and providing a new approach for cancer treatment.

[0064] Example 3 Experimental Data Supporting Explanation:

[0065] Experimental materials:

[0066] Control group: Using existing technology (intron self-cleavage method), the circularized sequence was the wild-type T4 phage intron (SEQ ID NO.10), and the transcriptase was conventional T7 RNA polymerase (NEB).

[0067] Experimental group: The optimized scheme of this invention was adopted, the circularized sequence was a mutant type II intron (SEQ ID NO.3, containing 5 base mutations), and the transcriptase was a thermostable T7 polymerase.

[0068] Target RNA: CAR-T-encoding long RNA (2200nt, containing CD19 scFv sequence).

[0069] Detection method:

[0070] Circulation efficiency: Agarose gel electrophoresis (2% gel, 120V electrophoresis for 30 min) combined with ImageJ grayscale analysis was used to calculate the percentage of grayscale of circular RNA bands (closed circular structures have a higher migration rate than linear RNA) to total RNA.

[0071] Exogenous residues: Linear template DNA residues (primer targeting vector backbone sequence, amplified fragment 150bp) were detected by RT-qPCR, with GAPDH as an internal control, and the percentage of residues relative to the initial template was calculated.

[0072] Experimental replication: Each group was independently repeated 3 times. Results are expressed as mean ± standard deviation (x ± s). The t-test was used to analyze the significance (P < 0.05 was considered significant).

[0073] II. Experimental Results

[0074]

[0075]

[0076] Note: Stability testing is the percentage of remaining circular RNA relative to the initial amount after incubation at 37°C for 24 hours.

[0077] Results analysis:

[0078] Mechanism for improved cyclization efficiency: The mutant type II intron (SEQ ID NO.3) improves the self-cleavage efficiency from 65% to 94% of the wild type by optimizing the 5' splice site (GU→AU) and the branch point adenosine (A→C) (verified by in vitro splicing experiments). Combined with the continuous synthesis ability of the thermostable T7 polymerase (2-fold increase in elongation rate), the cyclization efficiency exceeds 90%.

[0079] The reason for the reduction in exogenous residue is that this invention introduces a double enzyme digestion step (restriction endonucleases EcoRI and HindIII, with recognition sites located outside the circularized sequence) after circularization. The linear DNA template is specifically cleaved into fragments of <100bp, and after digestion with RNase-free DNase I (Thermo Fisher), the residual amount is reduced to below 5%.

[0080] Stability enhancement verification: Circular dichroism (CD) analysis showed that the proportion of double-stranded regions in the secondary structure of the circular RNA prepared in this invention was increased to 68% (compared to 45% in the control group), significantly enhancing resistance to RNase R (residual amount after 30 min digestion: 78% in the experimental group vs. 32% in the control group).

[0081] The optimized scheme of Example 3 of this invention (mutant circularized sequence + thermostable enzyme + double enzyme digestion purification) can significantly improve the preparation efficiency and purity of long circular RNA. The circularization efficiency is increased by 53.8% compared with the prior art (from 60.2% to 92.6%), the exogenous DNA residue is reduced by 72.4% (from 15.3% to 4.3%), and the product stability meets the clinical requirements for in situ expression of CAR-T cells (the half-life in vivo is extended by 1.5 times in 24h).

[0082] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing circular nucleic acids, characterized in that: It includes a transcription vector construction module (101), a circular sequence design module (102), an in vitro transcription module (105), a circularization module (103), and a purification module (104); The transcription vector construction module (101) constructs a transcription vector containing a promoter, a target gene, and a circularized sequence, with the promoter located upstream of the target gene; the circularized sequence design module (102) designs a circularized sequence that is linked to the target gene in the transcription vector; the in vitro transcription module (105) generates a linear nucleic acid molecule using the transcription vector as a template; the circularization module (103) circularizes the linear nucleic acid molecule to form a circular nucleic acid; and the purification module (104) purifies the circularized product. The transcription vector construction module (101) is responsible for constructing a transcription vector containing a promoter, a target gene, and a circular sequence. The promoter is located upstream of the target gene and drives the transcription process. The circular sequence design module (102) designs a circular sequence to guide nucleic acid molecules to form a closed circular structure. The circular sequence is closely linked to the target gene in the transcription vector. The in vitro transcription module (105) is directly connected to the transcription vector construction module (101); The cyclization module (103) uses a specific enzyme or self-cleavage mechanism to cyclize linear nucleic acid molecules into circular nucleic acids. The cyclization module (103) receives linear nucleic acids generated by the in vitro transcription module and works in synergy with the cyclization sequence design module (102). The cyclization sequence guides the cyclization reaction. The purification module (104) removes impurities, unreacted raw materials and exogenous sequences from the reaction system to obtain high-purity circular nucleic acid. The purification module (104) is connected to the cyclization module (103).

2. The method for preparing a circular nucleic acid according to claim 1, characterized in that: In the transcription vector construction module (101), the promoter is the T7 promoter, which specifically binds to RNA polymerase to drive transcription initiation, and the promoter and the target gene are connected by complementary base pairing. In the transcription vector construction module (101), the vector is pUC19, and the vector and the sequence containing type II introns and the target gene are connected by ligase at 37°C for 2 hours.

3. The method for preparing circular nucleic acid according to claim 1, characterized in that: In the circularization sequence design module (102), the circularization sequence is selected from any one of SEQ ID NO.2-9, and the circularization sequence guides the nucleic acid molecule to form a closed loop through base complementary pairing and spatial conformation.

4. The method for preparing circular nucleic acid according to claim 1, characterized in that: In the in vitro transcription module (105), T7 thermostable RNA polymerase is used for transcription. This enzyme performs in vitro transcription at 37-52°C. The in vitro transcription module (105) and the transcription vector construction module (101) are connected through the transfer of the transcription vector.

5. The method for preparing circular nucleic acid according to claim 1, characterized in that: The cyclization module (103) performs cyclization using either a type II intron self-cleaving mechanism or a T4 RNA ligase method. If a type II intron self-cleaving mechanism is used, different domains of the type II intron cooperate to achieve self-cleavage and cyclization. If a T4 RNA ligase method is used, the T4 RNA ligase catalyzes the formation of a phosphodiester bond between the 5' phosphate group and the 3' hydroxyl group of the linear nucleic acid molecule to achieve cyclization. The cyclization module (103) receives the linear nucleic acid generated by the in vitro transcription module (105) and works synergistically with the cyclization sequence design module (102). In the circularization module (103), if the T4 RNA ligase method is used, the reaction is carried out overnight at 16°C. The T4 RNA ligase and the linear nucleic acid molecule are circularized by specifically binding to recognize the sequences at both ends.

6. The method for preparing a circular nucleic acid according to claim 1, characterized in that: The purification module (104) uses column chromatography or a continuous flow chromatography system to purify the reaction system, removing impurities, unreacted raw materials and exogenous sequences. The purification module and the cyclization module are connected through the transfer of cyclization products. In the purification module (104), when column chromatography is used for purification, the circular nucleic acid and impurities are separated and purified according to their different physicochemical properties.

7. The method for preparing a circular nucleic acid according to claim 1, characterized in that: The prepared circular nucleic acids are circular RNA or circular DNA. When circular RNA is prepared, it can be used for vaccine antigen expression or CAR-T encoding; when circular DNA is prepared, it can be used for miRNA sponges or diagnostic probes.

8. The method for preparing a circular nucleic acid according to claim 1, characterized in that: For large-scale preparation, a 10L reaction system was used. The vector was prepared in batches by culturing E. coli containing the transcription vector in a fermenter and extracting the vector DNA by alkaline lysis. Automated circularization was carried out using a bioreactor and the reaction conditions were controlled by an automated control system. Continuous purification was carried out using a continuous flow chromatography system. The purity of the prepared circular nucleic acid was ≥95% and the endotoxin was ≤0.1EU / mL.

9. The method for preparing a circular nucleic acid according to claim 1, characterized in that: In preparing circular RNA for lung cancer vaccines, the vector construction involved inserting a sequence containing type II introns and the lung cancer antigen gene into the pUC19 vector. In vitro transcription was performed at 37°C for 3 hours, followed by circularization at 37°C for 2 hours. Purification was carried out using column chromatography.

10. The method for preparing a circular nucleic acid according to claim 1, characterized in that: When preparing circular DNA for liver cancer treatment, the linear DNA was designed to contain the SEQ ID NO.1 sequence that can bind complementary to the oncogenic miRNA. Enzymatic circularization was performed using T4 RNA ligase overnight at 16°C, and RNase R digestion was carried out at 37°C for 1 hour.

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