Plasmid vector for self-circularization RNA synthesis

Abstract

The present invention relates to a plasmid vector for synthesizing a self-circularization RNA structure in an in vitro transcription (IVT) process and to a DNA fragment for preparing same. The present invention can be used in a method for efficiently and economically producing circular RNA in large quantities under an STS system.

Description

Plasmid vector for self-circularizing RNA synthesis

[0001] The present invention relates to a DNA fragment inserted into a plasmid for synthesizing an RNA structure that is self-circularized during an in vitro transcription (IVT) process.

[0002] Circular RNAs (circRNAs) are covalently linked, single-stranded transcripts. Tens of thousands of circRNAs have been identified across diverse organisms using RNA-seq data and newly developed bioinformatics approaches. In eukaryotes, circRNAs are generated from mRNA through back-splicing and are known to function as microRNA sponges in vivo, regulating gene expression.

[0003] CircRNAs with open 5' and 3' ends exist very stably in vivo due to their structural characteristics, and many studies are currently being conducted on methods to produce genes for expression in the form of circRNAs.

[0004] Group Ⅰ intron ribozyme can induce trans-splicing by linking separately existing transcripts at the 3' end of the cleaved target RNA through two consecutive trans-esterification reactions. The STS (self-targeting & splicing reaction) system and the PIE (permuted intron exon) system are both used as methods to produce circRNA using Group Ⅰ intron ribozymes. The PIE system is a method to insert a target gene between the 3' intron fragment and the 5' intron fragment of the Group Ⅰ intron ribozyme to produce circRNA containing the target gene, and the STS system is a method to produce circRNA containing the target gene by designing the target gene to be located downstream of the Group Ⅰ intron ribozyme without cleavage.

[0005] The production of long circular RNA precursors using PCR suffers from high costs and low efficiency. Therefore, the inventors of the present invention have conducted extensive research into the design of an optimal plasmid DNA vector for self-circularizing RNA expression to mass-produce circular RNA using an STS system, and have completed the present invention.

[0006] [Prior Art Literature]

[0007] [Patent Document]

[0008] WO 2019 / 236673

[0009] KR 10- 2442946

[0010] The technical problem to be achieved by the present invention is to provide a plasmid vector for synthesizing a self-circularizing RNA structure, a DNA fragment used in producing the plasmid vector, and a method and system for designing a template for the DNA fragment.

[0011] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0012] To solve the above problem, the present invention provides a DNA fragment to be inserted into the production of a plasmid for synthesizing a self-circular RNA structure.

[0013] In the present invention, the DNA fragment refers to a DNA fragment in which a polynucleotide comprising a template including a gene sequence encoding a self-circularizing RNA structure and a sequence complementary thereto forms a double helix through hydrogen bonding.

[0014] In the present invention, the self-circularizing RNA structure has the following structure:

[0015] 5' - IGS (internal guide sequence) - Ribozyme - gene of interest - target site - 3'

[0016] In the present invention, the IGS region forms a guanine (G): uracil (U) wobble base pair with the target site, and the guanine forming the wobble base pair is located at the 5' end of the IGS region, and the uracil forming the wobble base pair is located at the 3' end of the target site region.

[0017] In the present specification, the uracil base included at the 3' end of the target site region may be indicated as a target splicing site as a position trans-spliced ​​by a ribozyme.

[0018] The DNA template included in the DNA fragment of the present invention is a DNA template having a restriction enzyme recognition site sequence linked to the 3' end of a gene encoding self-circularizing RNA, and the restriction enzyme recognition site sequence may not be included in the base sequence within the region encoding self-circularizing RNA.

[0019] In the present invention, the ribozyme is a Group Ⅰ intron ribozyme.

[0020] As one embodiment of the present invention, the restriction enzyme may be one selected from the group consisting of Swal, BplI, AjuI, PsrI, BcgI, and FalI.

[0021] As another embodiment of the present invention, the target site region may overlap with the target gene region.

[0022] As another embodiment of the present invention, the base sequence of the IGS region may be reverse complementary to the base sequence of the region of the target site, excluding the guanine.

[0023] As another embodiment of the present invention, the self-circularizing RNA structure may include nucleotides extending in the 5' direction of the IGS region to form a P10 helix.

[0024] As another embodiment of the present invention, the self-circularizing RNA structure may include nucleotides extending in the 5' direction of the IGS region and the 3' direction of the target site to form a P1 helix.

[0025] As another embodiment of the present invention, the self-circularizing RNA structure may not form a P10 helix.

[0026] As another embodiment of the present invention, the self-circularizing RNA structure may further include an AS (antisense sequence) region in the 5' direction of the IGS region and an ABS (antisense binding sequence) region capable of complementarily binding to the AS region in the 3' direction of the target site.

[0027] As another embodiment of the present invention, the self-circularizing RNA structure may be a ribozyme region and a target gene region or a target gene and a target site region connected by a spacer region consisting of a random base sequence.

[0028] As another embodiment of the present invention, the DNA template may additionally include a promoter upstream of a gene encoding a self-circularizing RNA.

[0029] As another embodiment of the present invention, the gene encoding the self-circularizing RNA and the promoter may be directly linked.

[0030] In addition, the present invention provides a plasmid vector into which the above DNA fragment is inserted.

[0031] The above plasmid may be a known or commercially available plasmid vector.

[0032] In addition, the present invention provides a transformed cell containing the plasmid vector, and the cell may be E. coli.

[0033] In addition, the present invention provides a method for producing a DNA template capable of synthesizing a self-circularizing RNA in an in vitro transcription (IVT) reaction, comprising the steps of: culturing the transformed cell; isolating and purifying the plasmid vector from the cell; and treating the isolated and purified plasmid vector with a restriction enzyme that recognizes and cuts a restriction enzyme recognition site designed to be linked to a gene encoding a self-circularizing RNA.

[0034] In addition, the present invention provides a method for designing insert DNA used in the production of a plasmid vector for self-circularizing RNA synthesis, comprising the following steps:

[0035] 5' - IGS (internal guide sequence) - Ribozyme - gene of interest - target site - 3' structure of self-circularizing RNA; Step of acquiring base sequence data of the gene;

[0036] A step of confirming a restriction enzyme recognition site sequence present within the base sequence of the above gene; and

[0037] A template design step of an inset DNA in which a restriction enzyme recognition site sequence, excluding the restriction enzyme recognition site sequence present within the base sequence of the above gene, is directly connected to the 3' end of the above gene.

[0038] In addition, the present invention provides a client into which a base sequence of a target gene is input;

[0039] A memory storing one or more restriction enzyme recognition site sequence data;

[0040] A first computation unit that is connected to the client and generates base sequence data of a gene encoding a self-circularizing RNA having a structure of 5' - IGS (internal guide sequence) - Ribozyme - gene of interest - target site - 3';

[0041] A second operation unit connected to the first operation unit and the memory, receiving a restriction enzyme recognition site sequence from the memory, and determining whether the restriction enzyme recognition site sequence is included in the base sequence of the first operation unit;

[0042] Provided is an insert DNA design system for producing a plasmid vector for synthesizing self-circular RNA, which includes an output unit that generates DNA sequence data in which the restriction enzyme recognition site sequence is linked to the 3' end of a gene encoding self-circular RNA when the restriction enzyme recognition site sequence is not included in the base sequence of the first operation unit.

[0043] In producing a DNA template for self-circularizing RNA synthesis, the present invention utilizes a plasmid vector, enabling the economical production of large quantities of DNA templates compared to PCR. Furthermore, the DNA template produced using the plasmid vector designed in the present invention can synthesize transcripts exhibiting circularization efficiencies equivalent to or greater than those of P1 constructs synthesized by PCR under in vitro transcription (IVT) reactions.

[0044] Figure 1 is a schematic diagram of a self-circularizing RNA structure and a circular RNA generated after the structure and STS reaction thereof.

[0045] Figure 2 is a schematic diagram of a process for producing circular RNA using a plasmid DNA vector, showing the production of a DNA template for expressing a self-circular RNA structure by treating a plasmid DNA vector with a restriction enzyme, the self-circular RNA structure produced as a result of performing IVT using the DNA template, and the circular RNA formed as a result of the STS reaction thereof.

[0046] Figure 3 is the sequence information of the P1 structure used in the experiment.

[0047] Figure 4 shows information on the plasmid vector used in the experiment.

[0048] Figure 5 is a schematic diagram of a DNA template design to be inserted into a plasmid vector, specifically showing a portion of a plasmid vector into which a DNA template with an added SwaI recognition site is inserted and the location of the restriction enzyme recognition site included therein.

[0049] This is a drawing showing the level of circular RNA production confirmed through PAGE after performing IVT on a DNA template for self-circular RNA expression obtained by treating the plasmid vector of FIG. 6 with SwaI, EcoRV, or NotI.

[0050] Figure 7 is a diagram showing the level of circular RNA production confirmed through PAGE after performing IVT on a plasmid DNA template obtained by treating SwaI and a PCR DNA template imitating the same.

[0051] Figure 8 is a schematic diagram of a DNA template design to be inserted into a plasmid vector, specifically showing a portion of a plasmid vector into which a DNA template with an added BplI recognition site is inserted and the positions of the restriction enzyme recognition sites included therein.

[0052] Figure 9 is a drawing showing the level of circular RNA production confirmed through PAGE after performing IVT on a DNA template for self-circular RNA expression obtained by treating the plasmid vector of Figure 8 with BplI.

[0053] Figure 10 is a restriction enzyme recognition site sequence applicable to the production of a DNA template used for producing a plasmid vector for self-circularizing RNA expression.

[0054] Figure 11 is a drawing showing the level of circular RNA production confirmed through PAGE after performing IVT on a DNA template for self-circular RNA expression obtained by treating each restriction enzyme with a plasmid vector for self-circular RNA expression to which an AjuI or PsrI recognition site has been added.

[0055] In a previous study, the present inventors developed a system (hereinafter referred to as a 'Circularization system by self-targeting & splicing reaction') in which an RNA structure carrying a target gene is circularized by performing self-targeting and splicing reaction using a trans-splicing ribozyme (T / S ribozyme).

[0056] In the present invention, the self-circularizing RNA structure has the following structure as a precursor of circular RNA:

[0057] 5' - IGS (internal guide sequence) - Ribozyme - gene of interest - target site - 3'.

[0058] The above IGS region forms a guanine (G): uracil (U) wobble base pair with the target site, and the guanine forming the wobble base pair is located at the 5' end of the IGS region, and the uracil forming the wobble base pair is located at the 3' end of the target site region.

[0059] More specifically, the IGS region is composed of a base sequence of 5'-GNNNNN-3', the target site region is composed of a base sequence of 5'-N'N'N'N'N'U-3', and N of the IGS region and N' of the target site region can each independently be A, G, C, or U, but preferably, at least one nucleotide can be a nucleotide to which the IGS region and the target site region can complementarily bind, and preferably, the base sequence of the IGS region can be reverse complementary to the base sequence of the region of the target site, excluding the guanine.

[0060] Meanwhile, in the present invention, the target site region is a region containing bases spliced ​​by a ribozyme, and may overlap with the target gene region depending on the base sequence design of the IGS region. Even in this case, the target site within the target gene is named separately. That is, in the present invention, the target site may overlap with part or all of the target gene region, or may exist separately from the target gene.

[0061] Meanwhile, the P1 helix refers to a helix structure formed through complementary binding of the target site by the IGS and the ribozyme during the formation of the secondary structure of the group I intron ribozyme, and the P10 helix refers to a helix structure formed through complementary binding of the shear region of the ribozyme and the 5'-direction base sequence of the transcript to be cleaved by the ribozyme.

[0062] In the present invention, the self-circularizing RNA structure may include nucleotides extending in the 5' direction of the IGS region to form a P1 helix and / or a P10 helix, wherein the P1 helix is ​​formed in a region where complementary binding between the IGS region and the target site occurs together with nucleotides extending in the 3' direction of the target site, and the P10 helix may be formed in a region where nucleotides extending in the 5' direction of the IGS region complementarily bind to a sequence that is reverse complementary to the extended nucleotides located between the ribozyme and the GOI region. The length of the extended nucleotides forming the P1 helix may be 3 nt, and the length of the extended nucleotides forming the P10 helix may be 6 nt.

[0063] Additionally, the self-circularizing RNA structure may include an antisense sequence (AS) region and an antisense binding sequence (ABS) region that can complementarily bind to each other at the 5' end and the 3' end.

[0064] In addition, the self-circularizing RNA structure may additionally include a spacer region consisting of a random base sequence, and the spacer region may be located between the IGS region and the target gene region and / or between the target gene region and the target site region. The spacer region may include or consist of poly(A), and poly(A) may be a polynucleotide in which adenine (A) is repeatedly linked, and the A may be linked by repeating 10 to 50 times.

[0065] Additionally, the target gene may or may not include a gene encoding a protein, but if the target gene includes a gene encoding a protein, the target gene may be designed to include an IRES (internal ribosome entry site) sequence.

[0066] In the present invention, when the target gene comprises a protein-encoding gene, the IRES in the circular RNA generated by the STS reaction of the self-circularizing RNA structure is designed to be located upstream of the protein-encoding gene. In this case, the protein-encoding gene for expression is designated as the target gene, and the IRES is designated separately from the target gene.

[0067] In the present invention, the self-circularizing RNA construct can be designed to minimize the immunogenicity exhibited by the resulting circular RNA in vivo. For example, a circular RNA intended for protein expression may contain only the IRES and target gene (protein-coding gene) sequences.

[0068] A self-circularizing RNA structure that forms a circular RNA containing only the IRES and the target gene sequence can be designed using the following methods. (1) If the 3' end of the target gene is U (uracil), the target site is designed to overlap with the target gene, and the IGS is designed accordingly. (2) If the base at the 3' end of the target gene is not U, an appropriate target site is selected on the target gene sequence, and the target gene is divided into a 5' fragment and a 3' fragment before and after the target splicing site within the selected target site, and the positions of the 5' fragment and the 3' fragment containing the target splicing site are reversed and designed. In this case, the conformation of the target gene that was intended to be loaded into the original circular RNA is preserved in the circular RNA generated by trans-splicing at the target splicing site. As an example, the P1 structure is presented in Fig. 3. In Fig. 3, the P1 construct is designed by selecting an appropriate target site on the IRES sequence and separating the IRES before and after the selected target site so that it is positioned at the 5' end and 3' end of the target gene, respectively. In this case, a fully functional IRES is positioned upstream of the target gene on the circular RNA generated by trans-splicing at the target site.

[0069] For mass production of circular RNA under the STS system, it is more cost-effective and efficient to mass-produce and purify plasmid DNA templates using microorganisms such as E. coli and obtain them through restriction enzyme reactions rather than using PCR templates.

[0070] Accordingly, the present inventors attempted to design a plasmid vector for producing a DNA template for synthesizing the self-circular RNA structure described above.

[0071] A promoter is required for the transcription of a self-circularizing RNA construct. In this specification, a construct comprising a gene encoding the self-circularizing RNA described above and a promoter operably linked to the gene is referred to as a "P1 construct."

[0072] The P1 construct is a construct designed so that its expression product (i.e., self-circularizing RNA construct) skips the first transesterification and only undergoes the second transesterification, thereby improving the efficiency of the transsplicing reaction. Thus, a P1 construct can be produced through PCR in which the gene of the target site ends precisely at the 3' end. However, depending on the position of the restriction enzyme recognition site in order to produce a DNA template for self-circularizing RNA synthesis using a plasmid vector, an unnecessary RNA sequence may remain after the target site of the self-circularizing RNA construct produced after run-off transcription. Due to this unnecessary RNA sequence, the first transesterification reaction is essential for self-circularization. It was predicted that the self-circularizing RNA construct that essentially requires the first transesterification reaction would have a lower circular RNA production efficiency than the transcript of the P1 construct that only requires the second transesterification reaction.

[0073] To verify the above hypothesis, the present inventors constructed a plasmid vector by adding a restriction enzyme recognition site sequence to the 3' end of the P1 construct, and compared the P1 construct and the IVT reaction under conditions in which the bases added in the 3' direction of the site where the splicing reaction occurs by the ribozyme were restricted and not. As a result, it was found that the more bases added in the 3' direction of the splicing reaction occurs, the lower the self-circularization efficiency.

[0074] Accordingly, the present inventors provide a DNA fragment to be inserted into a plasmid for producing a DNA plasmid vector for synthesizing a self-circularizing RNA structure, wherein the DNA template in the DNA fragment may be a restriction enzyme recognition sequence linked to the 3' end of a gene encoding the self-circularizing RNA structure described above, and preferably, the gene encoding the self-circularizing RNA structure and the restriction enzyme recognition site sequence may be directly linked.

[0075] That is, the present inventors intend to effectively produce a DNA template capable of synthesizing a self-circularizing RNA structure under IVT using a plasmid capable of independent replication within a cell as a vector, and the present invention intends to design a DNA fragment (insert DNA) to be inserted into the plasmid vector to improve the circularization efficiency of the self-circularizing RNA structure synthesized from the DNA template, and to provide the DNA fragment, a plasmid vector into which the DNA fragment is inserted, and a method for designing the DNA fragment.

[0076] In this specification, a DNA template refers to a polynucleotide used for self-circularizing RNA synthesis during IVT. However, in the production of the plasmid vector of the present invention, the DNA template is inserted into the plasmid vector in the form included in a DNA fragment (insert DNA), and in the DNA fragment of the present invention, the DNA template is provided by being designed with a structure in which a restriction enzyme recognition sequence is added, so that the DNA template included in the DNA fragment in the present invention refers to a polynucleotide in which a restriction enzyme recognition sequence is linked to the 3' end of a gene encoding a self-circularizing RNA structure. Therefore, in this specification, 1) a polynucleotide used for self-circularizing RNA synthesis and 2) a polynucleotide used for plasmid vector production can both be expressed as DNA templates, but they are interpreted with different meanings depending on whether it is an IVT reaction step or a plasmid vector production step.

[0077] Meanwhile, it is preferable that the restriction enzyme recognition site sequence used to produce the DNA template included in the insert DNA does not exist within the self-circularizing RNA structure.

[0078] In this specification, a DNA fragment inserted into a plasmid during the production of a plasmid vector is referred to as “insert DNA” or “DNA fragment,” and the DNA fragment is provided as a double-stranded DNA fragment formed by hydrogen bonding between a designed DNA template and a complementary sequence thereof.

[0079] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0080] [Experimental Method]

[0081] 1. P1 structure design

[0082] The P1 construct used in the experiment was designed as shown in Fig. 3. The P1 construct capable of inducing self-cyclization of the GOI, CVB3-sGFP, using the AU11 target site present in CVB3 IRES was used.

[0083]

[0084] 2. pTOP V2 vector

[0085] The pTOP V2 vector from Engenomics was used to deliver the DNA template for self-circularizing RNA expression into E. coli. As described in the Engenomics product manual, when the DNA template is inserted by TA cloning (when inserted in the forward direction), various restriction enzyme cleavage sites, such as EcoRV and NotI, are available at the back of the DNA template for generating the P1 construct (Fig. 4).

[0086]

[0087] 3. Design of DNA template with inserted Swal recognition site

[0088] When the DNA template for run-off transcription is created by cutting the DNA with EcoRV and NotI, which are restriction enzyme sites that exist only once after the target site in the pTOP vector, a 19-nt sequence is added from the target splicing site to the antisense strand used as a template for RNA synthesis in the case of EcoRV, and a 38-nt sequence is added in the case of NotI.

[0089] As an example, in the case of the AU11 target site (TTTATT) presented, if only TAAAT is added at the end, RNA with only one base sequence added at the target splicing site can be created using the SwaI restriction enzyme. Therefore, plasmid DNA was prepared by synthesizing and cloning insert DNA with the TAAAT sequence added, as shown in Fig. 5.

[0090]

[0091] 4. Design of DNA template with BplI recognition site inserted

[0092] Using gBlock for insertion of the BplI recognition site, insert DNA with the BplI recognition site linked to the P1 structure was designed and produced (Fig. 8).

[0093]

[0094] 5. Preparation of DNA template for self-circular RNA expression

[0095] A plasmid vector for self-circular RNA expression was constructed by inserting a DNA template designed by TA cloning into the pTOP vector using the TOPcloner TA-Blunt kit (Engenomics). The constructed vector was transformed into DH5alpha Escherichia coli, and the E. coli was selected and cultured according to the protocol recommended by Engenomics, after which the plasmid was purified.

[0096] Plasmid DNA was produced in large quantities in E. coli, extracted, and then single digested using each restriction enzyme, electrophoresed on an agarose gel, and purified using a gel extraction kit (Cosmogene Tech) for use in IVT.

[0097] If a restriction enzyme recognition site exists in the P1 structure, a plasmid DNA template for run-off transcription is produced using a restriction enzyme excluding the site.

[0098] All restriction enzymes used in this experiment are commercially available.

[0099] In the following experiments, the DNA template for self-circular RNA expression obtained by treating a plasmid vector with a restriction enzyme is called a 'plasmid DNA template', and the one produced using PCR is called a 'PCR DNA template'.

[0100]

[0101] 6. Confirmation of circular RNA production after IVT: PAGE

[0102] IVT reaction conditions were as follows (NEB's HiScribe T7 High Yield RNA Synthesis Kit and the provided protocol were used, but general in vitro transcription reaction kits can be used):

[0103] After 3 hours of reaction at 37°C in a 20 uL scale (1 ug T7 DNA template, 1 X Reaction buffer, 10 mM each ATP, UTP, CTP, GTP, T7 RNA polymerase mix 2 ul), 29 uL of nuclease-free water was added, and then 1 uL of RNase-free DNase I (10 U / ul) was added and reacted at 37°C for 30 minutes.

[0104] Column purification was performed using the Monarch RNA cleanup kit (NEB). Concentration was measured using a Nanodrop (Thermo Fisher Scientific) device.

[0105] Each 200 ng sample was mixed well with 10 M Urea-BPB (1X TBE) dye at a minimum sample:Dye=1:1 or higher dye ratio, heated at 75°C for 5 minutes, and analyzed by 4% Polyacrylamide-7 M Urea denature PAGE (electrophoresis at 50 W for 2 hours while maintaining the temperature at 50°C) (Gel was stained with SYBR Gold Nucleic Acid Stain product (Thermo Fisher Scientific) and analyzed using ImageQuant 800 (Cytiva product).

[0106]

[0107] [Experimental Results]

[0108] 1. Confirmation of self-cyclization efficiency according to the length of base added to the 3' end of the P1 structure.

[0109] The SwaI recognition sequence was added to the 3' end of the P1 construct and inserted into pTOP V2. The plasmid vector was treated with SwaI, EcoRV, or NotI restriction enzymes to obtain a DNA template for self-circular RNA expression, and the degree of circular RNA production was compared by performing IVT and PAGE. As a control, a P1 construct that was precisely terminated at the target site by PCR was used.

[0110] The DNA template obtained by treating with EcoRV has 19 nt bases added in the 3' direction from the target splicing site, and the DNA template obtained by treating with NotI has 38 nt bases added.

[0111] Run-off transcription samples obtained by digesting plasmids with EcoRV or NotI restriction enzymes all exhibited low self-circularization efficiencies. In contrast, DNA templates obtained by treatment with SwaI exhibited high self-circularization efficiencies, similar to those of the P1 construct (Fig. 6).

[0112]

[0113] 2. Comparison of PCR DNA template and plasmid DNA template

[0114] A template with the same sequence as the DNA template obtained by SwaI treatment was produced using PCR. The level of circular RNA produced was confirmed by performing PAGE after IVT using the plasmid DNA template obtained by SwaI treatment and the PCR DNA template. As a result, it was confirmed that DNA templates with an additional 1 nt base in the 3' direction from the target splicing site could produce high levels of circular RNA, regardless of the production method (Fig. 7). This suggests that the efficiency of circular RNA production is determined not by differences in the DNA template production method (PCR or restriction enzyme reaction of plasmid DNA), but by its design and sequence.

[0115]

[0116] 3. Use of various restriction enzymes

[0117] 3-1. A plasmid vector containing a DNA template with an added BplI recognition site was constructed, and the DNA template for self-circular RNA expression was prepared by restriction enzyme treatment (Fig. 8). There are two cleavage sites at the RE site. Even if partial digestion occurs with a low probability, where only the right cleavage site is cleaved and the left cleavage site is not cleaved, the RNA behind the target site will be cleaved and removed by the first transesterification reaction of the Group I intron in the produced RNA structure, so it is expected that there will be no problem in specifically producing the desired circular RNA.

[0118] The degree of self-circularization was compared by performing IVT using plasmid DNA templates cut with SwaI restriction enzyme and plasmid DNA templates cut with BplI restriction enzyme, which were previously prepared. As a result, the self-circularization efficiency was comparable or higher when cut with BplI compared to when cut with SwaI (Fig. 9).

[0119]

[0120] 3-2. Various restriction enzymes capable of precise cleavage without additional sequences after the target site were explored (Fig. 10). Among these, the same experiment as the previous experiment was performed using the AjuI and PsrI recognition sites. As a result, it was confirmed that the DNA template for self-circularizing RNA expression obtained by treating the inserted plasmid vector with each of the above restriction enzymes by adding the AjuI or PsrI recognition site in the 3' direction of the P1 structure produced a high level of circular RNA (Fig. 11). More specifically, in the case of PsrI, self-circularizing efficiency was shown to be equal to or higher than that of the case of cleavage with BplI. Meanwhile, AjuI showed a slightly lower self-circularization efficiency. In theory, if it properly cuts the RE site, there is no reason why it should be different from the DNA template made using other restriction enzymes, so it seems that the AjuI enzyme had a different DNA cutting efficiency compared to other restriction enzymes. As shown in the figure showing the RE site, both RE sites should be cut or at least the left RE site should be completely cut. However, it seems that due to the low activity of the AjuI enzyme, products that only cut the right RE site were mixed, creating a DNA template in which the target site was not located at the end, and this resulted in 1 st It is interpreted that the efficiency has decreased because a transesterification reaction is required.

[0121]

[0122] From the above, it was confirmed that circular RNA can be efficiently produced by self-circularization during the IVT process through vector design and restriction enzymes selected so that the final product of self-circularization structural RNA ends precisely at the target site using a plasmid DNA template that can be mass-produced without using PCR.

[0123]

[0124] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0125] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

Claims

1. A DNA fragment inserted into the production of a plasmid for synthesizing a self-circular RNA structure. In the above DNA fragment, the template is a restriction enzyme recognition site sequence directly linked to the 3' end of a gene encoding self-circularizing RNA. The above self-circularized RNA has a structure of 5' - IGS (internal guide sequence) - ribozyme - gene of interest - target site - 3'. The above IGS region forms a guanine (G): uracil (U) wobble base pair with the target site, The guanine forming the above wobble base pair is located at the 5' end of the IGS region, The uracil forming the above wobble base pair is located at the 3' end of the target site region, The above ribozyme is a Group Ⅰ intron ribozyme, A DNA fragment wherein the above restriction enzyme recognition site sequence does not overlap with a base sequence within a region encoding the self-circularizing RNA.

2. In paragraph 1, A DNA fragment, wherein the above restriction enzyme is one selected from the group consisting of Swal, BplI, AjuI, PsrI, BcgI, and FalI.

3. In paragraph 1, The above target site region is a DNA fragment that overlaps with the target gene region.

4. In paragraph 1, A DNA fragment characterized in that the base sequence of the IGS region is reverse complementary to the base sequence of the region of the target site, excluding the guanine.

5. In paragraph 1, The above structure is a DNA fragment that forms a P10 helix including nucleotides extending in the 5' direction of the IGS region.

6. In paragraph 1, A DNA fragment wherein the structure comprises nucleotides extending in the 5' direction of the IGS region and in the 3' direction of the target site, forming a P1 helix.

7. In paragraph 6, A DNA fragment wherein the above structure does not form a P10 helix.

8. In paragraph 1, The above self-circular RNA has an AS (antisense sequence) region in the 5' direction of the IGS region, A DNA fragment further comprising an ABS (antisense binding sequence) region capable of complementarily binding to the AS region in the 3' direction of the target site.

9. In paragraph 1, The above self-circularized RNA is a DNA fragment in which a ribozyme region and a target gene region or a target gene and a target site region are connected by a spacer region consisting of a random base sequence.

10. In paragraph 1, A DNA fragment wherein the above DNA template additionally includes a promoter upstream of a gene encoding a self-circularizing RNA.

11. In paragraph 10, A DNA fragment in which the gene encoding the self-circularizing RNA and the promoter are directly linked.

12. A plasmid vector into which the DNA fragment of paragraph 1 has been inserted.

13. In paragraph 12, The above vector is a plasmid vector, which is a pTOP vector. 14.5' - IGS (internal guide sequence) - Ribozyme - gene of interest - target site - 3' structure of self-circularizing RNA; Step of acquiring base sequence data of the gene; A step of confirming a restriction enzyme recognition site sequence present within the base sequence of the above gene; and A method for designing insert DNA used in the production of a plasmid vector for self-circularizing RNA synthesis, comprising a step of designing a template for an inset DNA in which a restriction enzyme recognition site sequence, excluding a restriction enzyme recognition site sequence present within the base sequence of the gene, is directly linked to the 3' end of the gene.

15. A client into which the base sequence of the target gene is input; A memory storing one or more restriction enzyme recognition site sequence data; A first computation unit that is connected to the client and generates base sequence data of a gene encoding a self-circularizing RNA having a structure of 5' - IGS (internal guide sequence) - Ribozyme - gene of interest - target site - 3'; A second operation unit connected to the first operation unit and the memory, receiving a restriction enzyme recognition site sequence from the memory, and determining whether the restriction enzyme recognition site sequence is included in the base sequence of the first operation unit; An insert DNA design system for producing a plasmid vector for synthesizing self-circular RNA, comprising an output unit that generates DNA sequence data in which the restriction enzyme recognition site sequence is linked to the 3' end of a gene encoding self-circular RNA when the restriction enzyme recognition site sequence is not included in the base sequence of the first operation unit.

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