KR20220061480A - Manufacturing method of circular nucleic acid template for producing high molecular weight protein using hydrogelated nucleic acid - Google Patents

Abstract

The present invention relates to a method for preparing a DNA double-stranded template for producing a high molecular weight protein, the method being capable of producing a protein hydrogel nucleic acid, and a high molecular weight protein production system using the same. The nucleic acid produced by the hydrogelated nucleic acid production system of the present invention overcomes the limitations of high cost and low yield in the existing intra/extracellular nucleic acid synthesis method, especially RNA synthesis, and stabilizes RNA due to hydrogelation, thereby being usefully used in the field of functional RNA-based technology. In particular, in terms of the production of proteins having a high molecular weight of 6 kDa or more in the existing G-quadruplex-based nucleic acid synthesis technology of the present inventors, the nucleic acid production system of the present invention overcomes the limitations of conventional mass-production of nucleic acid templates, so that nucleic acid templates for producing hydrogelated nucleic acids can be mass-produced at low cost. Furthermore, when producing peptides or proteins using the hydrogel nucleic acid system and the nucleic acid templates prepared by the method of the present invention, the production yield can be maximized. In particular, the same can be usefully used in a cell-free synthesis system for synthesizing peptides or proteins. Therefore, the same can be usefully used in the technology in various fields such as life science, medicine, and food based on the synthesis of functional peptides or proteins and functional nucleic acids.

Description

Manufacturing method of circular nucleic acid template for producing high molecular weight protein using hydrogelated nucleic acid and high molecular weight protein production system

The present invention relates to a method for producing a circular nucleic acid template for production of a high molecular weight protein capable of producing a protein hydrogelled nucleic acid and a high molecular weight protein production system using the same, and a nucleic acid comprising a G-quadruplex gene and a target sequence It relates to a method for producing a template and a system for producing a high molecular weight protein using the template.

Central dogma refers to a flow path of genetic information that was identified and named by British molecular biologist Francis Crick in 1956. DNA replication, DNA to RNA transcription , and RNA to protein translation. The initial concept meant the flow of expression from DNA to protein, but the current central dogma includes reverse transcription from RNA to DNA, and as various functions other than the protein coding function of RNA have been demonstrated, the function of genetic information and a concept that encompasses all flows. As research on the flow of genetic information and the function of the composition of the central dogma continues, the synthesis technology of genomes (DNA and RNA, etc.) and proteins (peptides) from the perspective of each stage of the central dogma has been developed in the life sciences, medicine, and alternative energy industries. It has been researched and developed as the most fundamental technology that has a significant impact on

In the existing gene synthesis technology, a method of synthesizing the precise nucleic acid sequence of a specific gene into short oligonucleotides and linking them is used. It is difficult to find errors of various factors that occur during nucleic acid sequencing and accurate sequencing analysis, and there are disadvantages in that only one type of gene of a specific nucleic acid sequence can be synthesized at a time (Mol Biosyst. 2009 Jul; 5 (Mol Biosyst. 2009 Jul; 5) 7):714-22.doi:10.1039/b822268c.Epub 2009 Apr 6.).

Techniques currently used for DNA synthesis mainly include PCR and typical plasmid purification procedures from bacterial and other cellular sources. Methods for purifying plasmids from bacteria and other cells are expensive in time/human/economical, and PCR, which is in vitro amplification, relies on rapid thermal cycling, which is impractical for large-scale use.

Functional single-stranded (eg mRNA) and double-stranded RNA molecules have been produced in living cells and in vitro using purified recombinant enzymes and purified nucleotide triphosphates (European Patent No. 1631675 and US Patent Application Publications). Double wide band (see No. 014/0271559 A1). Nevertheless, the production of RNA on a scale that allows for a wide range of commercial applications requires excessive costs. Recently, RNA-based development applications (e.g., siRNA or RNAzyme therapeutics or miRNA or piwi RNA-based diagnostic platforms, etc.) have been actively used in various fields, especially medicine, replacing existing DNA and proteins. According to the central dogma that sustains biology, it is produced from DNA to RNA to the final protein. RNA has a very short viability of a few seconds or minutes compared to DNA or protein, but it has a DNA genome code and a unique function of a protein at the same time, so its potential application is great. As such, the potential of RNA is illuminated, and various biological techniques for commercial use are plentiful, but there is a technically and costly very difficult in extracting RNA in cells or synthesizing it by chemical techniques. For example, since the degradation rate of intracellular RNA is faster than that of DNA, extraction or storage is difficult, and it is difficult to synthesize more than 50 bases in length with current chemical synthesis technology, and even if successful, the yield is very low. Current RNA production methods are divided into intracellular culture and chemical synthesis, both of which have limitations in spite of excellent applications of RNA due to high cost and low yield.

On the other hand, the G-quadraplex (G4) is a secondary structure of DNA found in the terminal region of chromosomes and transcriptional regulatory regions of various genes, and is divided into four It refers to a complex of G-quadrued strands formed in the form of hydrogen bonds by pairing DNA strands with each other. Telomeres are rich in guanine to form G-quadrules, which inhibit the activity of telomerase. Inhibition of the activity of telomerase using the characteristics of the G-quadrel has been proposed as a therapy for inducing apoptosis of tumor cells. In addition, it is known that the G-quadrel regulates the transcription of genes according to the formation site of the structure.

In order to solve the problems of the above-mentioned RNA synthesis (transcription) and protein production (translation) system and synthetic RNA, the present inventors use a nucleic acid containing a G-quadruplex motif sequence as a template for circular circular transcription (Rolling circle transcription: RCT)-based production of hydrogel nucleic acid and protein production system has been invented and a patent application has been filed (Korean Patent Application No. 10-2019-0136416, unpublished). Since the RNA synthesized by the nucleic acid production system is hydrogelled by forming a G-quartet structure, the stability of RNA can be significantly increased, and it is useful as a platform technology for cell-free protein synthesis from which peptides or proteins can be produced. can be used However, the method has a disadvantage in that hydrogelation is remarkably reduced when the target sequence is 150 bp or more or an unintentional secondary structure is formed. In general, since most genes encoding proteins used in various industries and medical fields have a long nucleotide sequence of 150 bp or more, such a significant reduction in hydrogelation has a disadvantage that makes it difficult to put the nucleic acid production system into practical use.

In order to overcome the above limitation, the present inventors added a double-wide band template having a short sequence of 30 to 100 bp or less, confirming that the nucleic acid is smoothly hydrogelled even when it has a long nucleotide sequence, and applied for a patent. (Korean Patent Application No. 10-2020-0077146, unpublished). However, in the original patent of No. 10-2019-0136416 and the double-wide band patent of No. 10-2020-0077146, the complex structure of the divided promoter, the sequence complementary to the target sequence, and the G-quadruplex sequence must be synthesized separately, After synthesizing in single-stranded form, there was the inconvenience of designing a primer sequence complementary to the promoter sequence and going through complicated processes such as annealing and ligation. Since single-stranded oligonucleotide templates of hundreds of bp to thousands of bp or more need to be synthesized for production, mass production is practically difficult.

Under this background, the present inventors have made intensive efforts to produce a template containing a sequence encoding a high molecular weight protein in the production system of the hydrogelled nucleic acid and protein production system. As a result, the target sequence and G- After cloning and amplifying a double-stranded DNA sequence containing a quadruplex motif, it is possible to mass-produce a circular nucleic acid template for the production of hydrogelled nucleic acids and high molecular weight proteins even at low cost through restriction enzymes or infusion cloning. , confirmed that it is possible to produce RNA hydrogel and high molecular weight protein in a cell-free protein synthesis system in high yield using the produced circular nucleic acid template, and completed the present invention.

The information described in the background section is only for improving the understanding of the background of the present invention, and it does not include information forming prior art known to those of ordinary skill in the art to which the present invention pertains. it may not be

It is an object of the present invention to provide a method for preparing a circular nucleic acid template for the production of hydrogelled nucleic acids.

Another object of the present invention is to provide a nucleic acid template prepared by the method for preparing the nucleic acid template.

Another object of the present invention is to provide a method for producing a hydrogelled nucleic acid and a hydrogelated nucleic acid prepared by the method.

Another object of the present invention is to provide a use of the hydrogelled nucleic acid.

Another object of the present invention is to provide a use for producing a peptide or protein of the hydrogelled nucleic acid.

In order to achieve the above object, the present invention provides (a) a promoter sequence; target sequence; and amplifying a nucleic acid strand comprising a promoter sequence, a target sequence, and a G-quadruplex sequence in a vector comprising a G-quadruplex seqeuence sequence; and

(b) converting the amplified nucleic acid strand into a circular nucleic acid template to obtain a circular nucleic acid template for producing hydrogelled nucleic acid.

The present invention is also prepared by the method for the preparation of the circular nucleic acid template for the preparation of the hydrogelled nucleic acid, and a sequence complementary to a promoter sequence; a sequence complementary to the target sequence; And it provides a circular nucleic acid template for preparing hydrogelled nucleic acids, characterized in that it comprises a G- quadruplex motif (G-quadruplex motif) sequence.

The present invention also provides a method for producing a hydrogelized nucleic acid comprising the step of amplifying or transcribed the circular nucleic acid template for preparing the hydrogelled nucleic acid.

The present invention is also prepared above, the promoter; target sequence; And it provides a hydrogelled nucleic acid comprising a G- tetramerization sequence.

The present invention also comprises the steps of preparing a hydrogelled RNA by transcription (transcription) of the circular nucleic acid template for preparing the hydrogelled nucleic acid; and

It provides a method for producing a peptide or protein comprising the step of translating the hydrogelled RNA, and synthesizing the peptide or protein.

The nucleic acid produced by the hydrogelation nucleic acid production system of the present invention overcomes the limitations of high cost and low yield in the existing intracellular/extracellular nucleic acid synthesis method, especially in the synthesis of RNA, and stabilizes RNA due to hydrogelation, so that functional RNA It can be usefully used in the base technology field. In particular, the nucleic acid production system of the present invention overcomes the limitation that it is difficult to mass-produce existing nucleic acid templates in the production of proteins having a high molecular weight of 6 kDa or more in the present inventors' G-tetraplex-based nucleic acid synthesis technology. Thus, it is possible to mass-produce a nucleic acid template for the production of hydrogelled nucleic acids even at low cost. Furthermore, when a peptide or protein is prepared using the nucleic acid template and hydrogelled nucleic acid system prepared by the method of the present invention, there is an advantage that the production yield can be maximized, and in particular, a cell-free synthesis system for synthesizing peptides or proteins can be usefully used for Therefore, it can be usefully used in various fields such as life science, medicine, food, etc. based on the synthesis of functional peptides or proteins, functional nucleic acids, and the like.

1 schematically shows a method for preparing a circular nucleic acid template for preparing a hydrogelled nucleic acid of the present invention.
Figure 1a: Development view of cloning green fluorescent protein (GFP) into an E. coli expression vector
Figure 1b: schematically shows a method for producing a circular nucleic acid template using the conjugation method. After PCR amplification from the T7 promoter sequence to the G-quartet, both ends were cut by treatment with restriction enzyme (BamHI), and then treated with ligase to ligate. Using T7 exonuclease, only circular nucleic acid template (circular DNA) was obtained and in vitro transcription was performed to synthesize RCT transcript.
Figure 1c: schematically shows a method for preparing a circular nucleic acid template using the infusion cloning method. After PCR amplification from the T7 promoter sequence to the G-quadruplex, the infusion cloning kit was processed and both ends were spliced. Using T7 exonuclease, only circular nucleic acid template (circular DNA) was obtained and in vitro transcription was performed to synthesize RCT transcript.
2 is a result confirming the formation of a circular nucleic acid template through 1% agarose gel electrophoresis.
Figure 2a: shows the results of the presence or absence of a purification process after treatment with BamHI. The PCR-amplified product of GFP was treated with BamHI to compare the presence or absence of the purification process, but it was confirmed that there was no significant difference on 1% agarose gel electrophoresis.
Figure 2b: It was confirmed that the purified product and the unrefined product were each ligated, showing the difference in ligation, and various complexes were formed in the purified product, whereas it was confirmed that a large amount of monomers were generated in the unrefined product did.
Figure 2c: As a result of T7 exonuclease treatment, it was confirmed that circular nucleic acid template (circular DNA) was not degraded only in the unpurified product.
3 confirms the reaction of T7 Exonuclease to linear and circular nucleic acid templates.
Figure 3d: As a result of confirming that T7 exonuclease degrades only linear DNA, it was confirmed that all of the PCR products (linear nucleic acid strands) were processed for 10, 20, and 30 minutes to be degraded.
FIG. 3e: As a result of confirming that T7 exonuclease degrades only linear DNA, it was confirmed that all of them were not degraded by treatment with a circular nucleic acid template (circular DNA) for 10, 20, or 30 minutes.
4 is a result confirming the formation of an RNA hydrogel by performing circular circulation transcription (RCT) using a circular nucleic acid template as a template through a 1% agarose gel electrophoresis image.
Figure 4f: A result of confirming the morphological difference of RNA transcripts synthesized using linear and circular nucleic acid templates as templates.
Figure 4g: The concentration of the RNA transcript according to the amount of the template nucleic acid template (linear and circular) was confirmed.
5 shows the results of preparing a circular nucleic acid template through infusion cloning using 1% agarose gel electrophoresis.
Figure 5a: A PCR product (linear nucleic acid) for infusion cloning was confirmed.
5B: It was confirmed that a circular nucleic acid template was generated through infusion cloning, and it was confirmed that various complexes were generated.
Figure 5c: Treated with T7 exonuclease to digest all of the linear DNA, and only the circular nucleic acid template was obtained.
5D: It is the result of confirming that the RCT transcript was produced by in vitro transcription of the obtained circular nucleic acid template.
6 is a result of verifying the GFP expression efficiency using the RNA hydrogel synthesized through RCT using 12% SDS-PAGE.
The information for each lane is as follows:
Lane 1: negative control, Lane 2: linear DNA, Lane 3: linear DNA+agarose, Lane 4: donut1(circular DNA1), Lane 5: donut1(circular DNA1)+agarose, Lane 6: donut2(circular DNA2), Lane 7: donut2 (circular DNA2) + agarose.
6A: A graph quantifying the fluorescence value (RFU) of green fluorescent protein produced after in vitro protein expression using RCT transcripts.
6b: A result of confirming the protein band of the produced green fluorescent protein through 12% SDS-PAGE electrophoresis.
7 shows GFP expression efficiency when using an RNA hydrogel generated by simultaneously performing in vitro transcription of a circular nucleic acid template (first nucleic acid template) and a double-wide band nucleic acid template (second nucleic acid template) for the preparation of hydrogelled nucleic acids. This is the verification result.
The information for each lane is as follows:
7a: Lane 1 (negative control): 168.46, Lane 2 (positive control): 4312.26, Lane 3: (GFP angel 30T-0.125 μM: 4614.99), Lane 4: (GFP angel 30T-0.25 μM: 4561.78), Lane 5: (GFP angel 30T-0.5 μM: 7257.58), Lane 6: (GFP angel 30T-0.75 μM: 7007.31), Lane 7: (GFP angel 30T-1 μM: 7185.96).
7b: Lane 1 (negative control): 168.46, Lane 2 (positive control): 4312.26, Lane 3: (GFP angel 60T-0.125 μM: 6320.14), Lane 4: (GFP angel 60T-0.25 μM: 6950.44), Lane 5: (GFP angel 60T-0.5 μM: 6132.26), Lane 6: (GFP angel 60T-0.75 μM: 7492.49), Lane 7: (GFP angel 60T-1 μM: 9491.85).
7c: Lane 1 (negative control): 168.46, Lane 2 (positive control): 4312.26, Lane 3: (GFP angel 90T-0.125 μM: 7075.71), Lane 4: (GFP angel 90T-0.25 μM: 7406.6), Lane 5: (GFP angel 90T-0.5 μM: 8556.68), Lane 6: (GFP angel 90T-0.75 μM: 9388.27), Lane 7: (GFP angel 90T-1 μM: 9658.04).
7d: Lane 1 (negative control): 168.46, Lane 2 (positive control): 4312.26, Lane 3: (GFP angel 120T-0.125 μM: 4486.84), Lane 4: (GFP angel 120T-0.25 μM: 6813.15), Lane 5: (GFP angel 120T-0.5 μM: 6666.26), Lane 6: (GFP angel 120T-0.75 μM: 8943.02), Lane 7: (GFP angel 120T-1 μM: 8098.43).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art. All nucleotide sequences described in this specification were written from the 5' end to the 3' end.

Currently, most of RNA is extracted and purified in cells, and with the current method, it is not easy to obtain an appropriate amount of RNA due to a fast degradation rate, and the post-purification process is very complicated, resulting in low yield and high cost.

Accordingly, the present inventors designed a nucleic acid template capable of producing a stable nucleic acid without a separate additional process by allowing the nucleic acid containing the target sequence to hydrogel through the automatic formation of the G-quadruplex formed in the living organism. In particular, by introducing it to a rolling circle amplification (RCA) or rolling circle transcription (RCT) system, the yield of RNA production was significantly increased, and furthermore, hydrogelled nucleic acid produced in a cell-free protein synthesis system After confirming that this remarkable protein expression rate and production yield were shown, the invention was completed and a patent application was applied. However, in the case of the nucleic acid template of the present invention, when the target sequence is a gene encoding a protein of 6 kDa or more, it is difficult to mass-produce a nucleic acid template having a complex sequence structure such as a promoter sequence and a G-quartet motif, which is isolated, and a high molecular weight protein There was a limit to applying the nucleic acid template to the production of

In order to overcome the above limitations, in one embodiment of the present invention, the present inventors introduced and amplified a nucleic acid having a relatively simple structure including a target sequence and a G-tetramerization sequence into a vector, and then a promoter sequence, a target sequence, And a nucleic acid template comprising a G-tetramerization sequence was amplified, and converted into a circular template using ligation or infusion cloning technology. It was confirmed that it is possible.

The nucleic acid template produced by the method of the present invention is characterized in that it includes a sequence complementary to a promoter sequence, a sequence complementary to a target sequence, and a G-quadruplex motif sequence. The nucleic acid template is described in detail in Korean Patent Application No. 10-2020-0077146 filed by the present inventors. Using the nucleic acid template as a template, a nucleic acid produced by performing amplification or transcription is hydrogelled to form a G-quartet to form a more stable structure.

In another embodiment of the present invention, it was confirmed that even when expressing a high molecular weight protein using the template prepared by the above method, it exhibited a significantly higher expression efficiency of about 4 to 6 times, and further, a double-wide band template was used. When used together, it was confirmed that an additional increase in protein expression efficiency of about 2 times was obtained.

Accordingly, the present invention, in one aspect, relates to a method for preparing a circular nucleic acid template for the production of hydrogelled nucleic acids. The method for producing a circular nucleic acid template for the production of hydrogelled nucleic acids of the present invention is

(a) a promoter sequence; target sequence; and amplifying a nucleic acid strand comprising a promoter sequence, a target sequence, and a G-quadraplex sequence in a vector comprising a G-quadraplex sequence; and (b) converting the amplified nucleic acid strand into a circular shape to obtain a circular nucleic acid template for producing hydrogelled nucleic acids.

The present invention overcomes the limitation that mass production is difficult with the existing method for preparing a nucleic acid template in the production of a protein having a high molecular weight of 6 kDa or more in the conventional G-quadrued-based nucleic acid synthesis technology of the present inventors, It has the advantage of being able to mass-produce a circular nucleic acid template for the production of hydrogelled nucleic acids even at low cost. Therefore, the prototype nucleic acid template for producing hydrogelled nucleic acids of the present invention is preferably used for the production of high molecular weight proteins.

Accordingly, in the present invention, when used for the production of a high molecular weight protein, the "prototype nucleic acid template for producing hydrogelled nucleic acid" is used interchangeably with the same meaning as "prototype nucleic acid template for producing a high molecular weight protein".

The "hydrogelled nucleic acid" of the present invention means a nucleic acid that has formed a gel through various interactions such as hydrogen bonding, covalent bonding, and hydrophobic interactions between nucleic acid sequences, and in the present invention, in particular, the G-tetramerization sequence is hydrogen It is characterized in that it forms a unique structure in the shape of a cube that forms a quadruple strand in the form of a bond to form a gel. In order to solve the above-described RNA synthesis method and the problem of synthetic RNA, the present inventors use a nucleic acid including a G-quadruplex motif sequence as a template. Based on rolling circle transcription (RCT) Invented and applied for a patent for a production system for hydrogelled nucleic acids of (Korean Patent Application No. 10-2019-0136416). Since the RNA synthesized by the nucleic acid production system is hydrogelled by forming a G-quartet structure, the stability of RNA can be remarkably increased, and it can be usefully used as a platform technology for producing peptides or proteins therefrom. . The "hydrogelled nucleic acid" may be used interchangeably with "a nucleic acid (eg, DNA or RNA) hydrogel".

In the manufacturing method of the present invention, the vector of step (a) may be introduced into a vector template through various methods or artificially synthesized and manufactured. In one embodiment of the present invention, by using restriction enzymes (NdeI and SalI) to clone the nucleic acid containing the target sequence and the G- tetramerization sequence into the multiple cloning site (MCS) of the pk7 vector containing the T7 promoter, the promoter sequence ; A vector including a target sequence and a G-tetramerization sequence was prepared, but the present invention is not limited thereto, and when the vector does not include a promoter, a nucleic acid strand comprising a promoter sequence, a target sequence and a G-tetramerization sequence is introduced. Vectors can also be made.

In the present invention, the promoter sequence; target sequence; and the G-tetramerization sequence may be introduced into the sense strand of the vector. In this case, a sequence complementary thereto may be introduced into the antisense strand of the vector.

In the present invention, the G-tetramerization sequence may be characterized in that it is linked to the 5' end or 3' end of the target sequence.

As used herein, the term "vector" refers to a carrier used to transport, replicate or express a specific nucleic acid sequence by recombination with a specific nucleic acid sequence (eg, a target sequence). Depending on the type of vector, it is divided into a plasmid vector, a viral vector, a cosmid vector, an artificial chromosome, etc., and it is divided into a cloning vector and an expression vector depending on the application. As the vector of the present invention, all types of vectors described above may be used without limitation.

In the present invention, the vector is used as a template for cloning and amplifying a nucleic acid template in large quantities. In the present invention, the vector may include an origin of replication, a restriction enzyme cleavage site, a multiple cloning site (MCS), and/or a selection marker. In the present invention, the vector may be characterized in that it contains a sequence necessary for gene expression, such as an expression vector. In the present invention, the vector may include an enhancer, a promoter sequence, a transcription initiation sequence, and/or a stop codon, and the like. In the present invention, the vector may be characterized in that it does not include a transcription termination sequence for continuous nucleic acid transcription. Examples of vectors that can be used in the present invention include, but are not limited to, pK7, pIVEX, pET, pTXB, pUC, pF3K, and the like.

As used herein, the term “promoter” refers to a gene region capable of regulating transcription initiation in the process of protein expression.

In the present invention, the promoter can be used by selecting a suitable promoter by those skilled in the art according to the type of template, the synthesis method, the type of nucleic acid to be produced, and the target sequence. In a double-stranded nucleic acid, a promoter is generally a concept including both a sense strand and an antisense strand, but in the present invention, for the distinction between both strands, a 'promoter sequence (sense sequence)' and a 'promoter sequence complementary sequence (antisense sequence) sequence)'.

In the present invention, the promoter is, for example, T7 promoter, T5 promoter, T3 promoter, lac promoter, tac and trc promoter, cspA promoter, lacUV5 promoter, Ltet0-1 promoter, phoA promoter, araBAD promoter, trp promoter, tetA There are promoters, Ptac promoters, SP6 promoters, and the like, and may preferably be selected from the above examples, but is not limited thereto.

As used herein, the term "target sequence" refers to a 'nucleic acid sequence to be produced (Sense)'. In the case of synthesizing DNA or RNA using the nucleic acid template as a template, a hydrogelled nucleic acid including a “nucleic acid to be produced” may be produced. The nucleic acid to be produced may be DNA, RNA, PNA, LNA, etc., preferably DNA or RNA. For example, the nucleic acid to be produced may be a functional moiety, and preferably may be an mRNA encoding a specific protein, but is not limited thereto.

In the present invention, when the circular nucleic acid template of the present invention is double-stranded, the target sequence may be included in the sense strand of the circular nucleic acid template for producing hydrogelled nucleic acid, and a sequence complementary thereto is the circular nucleic acid for producing hydrogelled nucleic acid. may be included in the antisense strand of the template.

As used herein, the term "G-quadruplex" refers to a structure in which two or more G-tetraplexes appearing in a DNA nucleotide sequence having a high ratio of guanosine residues are vertically stacked. , Here, G-quadrel refers to a unique cube-shaped DNA structure in which four guanosine-rich DNA strands are paired with each other to form a quadruple in a hydrogen bond form, and four guanine bases form a Hoogsteen hydrogen bond (Hoogsteen hydrogen bonding) refers to a rectangular structure formed densely. The G-quadrel is very stable under various biochemical conditions, and the orientation within the quaternary is variable. In particular, the cation (mainly K+) at the center of the G-quartet contributes to maintaining the structure of the G-quartet more stably. G-quadrules are known to have diverse biological functions in the telomere region and in the promoter region (Henderson E, et al., Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs., Cell (December 1987)). In the present invention, "G-tetramer" may be used interchangeably in the same meaning as "G-tetrapolymer".

G-quartets are formed predominantly at guanine-rich sequence sites (Murat P, Balasubramanian S (April 2014)). However, not all guanine-rich sequence sites form G-quartets. A method of predicting the ability to form a G-quadrel has been previously reported (Todd AK, Johnston M, Neidle S (2005)). For example, Todd AK, Johnston M et al. reported that d(G ₃₊ N _1-7 G ₃₊ N _1-7 G ₃₊ N _1-7 G ₃₊ ) (where the subscript is the range of numbers of each base, N is an arbitrary base, d is an integer greater than or equal to 1, meaning a repeat of the nucleotide sequence in parentheses) has been reported as a general pattern of forming a G-quadruplex. In the present invention, the G-quartet may be characterized in that it is formed by three or more consecutive guanines and a sequence in which 1 to 7 random sequences are repeated one or more times,

As used herein, the term “G-quadruplex sequence” refers to a sequence capable of forming a G-quadruplex, and the nucleic acid strand including the G-quadruplex sequence forms a G-quadruplex. make it possible The G-tetramerization sequence included in the nucleic acid strand transcribed using the nucleic acid template of the present invention as a template forms a G-quartet, thereby hydrogelation.

In the present invention, the G-tetramerization sequence may be characterized as a nucleic acid sequence in which 3 or more consecutive guanines and 1 to 7 random sequences are repeated one or more times, most preferably 5'-TAGGGTTAGGGT -3' (SEQ ID NO: 1).

As used herein, the term "G-quadruplex motif sequence" refers to any sequence capable of forming a G-quadruplex when amplified or transcribed.

In the present invention, the G-quadrel motif sequence may be characterized as complementary to the G-quartetization sequence, and when included in a double-stranded nucleic acid, the G_quartet motif sequence is the antisense strand of the vector. may be included.

In the present invention, preferably, the G-quartet motif sequence may be characterized in that it is complementary to a nucleic acid sequence in which 3 or more consecutive guanines and 1 to 7 random sequences are repeated one or more times, most preferably It may be characterized as 5'-ACCCTAACCCTA-3' (SEQ ID NO: 2).

In one embodiment of the present invention, the 5'-UAGGGUUAGGGU-3' sequence of RNA transcribed using the G-quartet motif of the antisense strand as a template was designed to form a G-quartet.

In the present invention, a G-tetramerization sequence may be included in the sense strand of a vector or nucleic acid template, and a G-tetramerization motif sequence may be included in the antisense strand of the vector or nucleic acid template.

Accordingly, the RNA transcribed using the circular nucleic acid template for the production of hydrogelled nucleic acids of the present invention as a template includes a G-tetramerization sequence, and may be characterized in that it is hydrogelled.

In the present invention, step (a) may be performed through various nucleic acid amplification methods. For example, the step (a) is a rolling circle amplification (Rolling Circle RCA), polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and transcription-mediated (TMA) amplification), and as in one embodiment of the present invention, preferably isothermal amplification using polymerase chain reaction (PCR), but is not limited thereto.

In the present invention, when amplifying a nucleic acid strand through PCR, the vector used for the amplification of step (b), the introduced nucleic acid sequence (target sequence, G-tetramerization sequence, etc.), the enzyme used (polymerase) , restriction enzymes, ligases, etc.) may be appropriately designed and used, and it is preferable that the amplified nucleic acid strand be designed and used so that it can include a promoter sequence, a target sequence, and a G-tetramerization sequence.

In an embodiment of the present invention, different primer pairs were designed, synthesized, and used according to the conversion method into a circular nucleic acid template.

In the present invention, a pair of primers may be used for amplification of a nucleic acid strand. In the present invention, the primer pair may be designed so that at least a promoter sequence, a target sequence, and a G-tetramerization sequence can be amplified.

In one embodiment of the present invention, when converting into a circular nucleic acid template using ligation, the same BamHI cleavage sequence is inserted at the 5' end of each primer in order to generate identical cleavage sites at both ends of the amplified nucleic acid strand. In addition, it was designed and manufactured.

Therefore, in the present invention, it may be characterized by including the same restriction enzyme cleavage site at the 5' end of the primer pair.

In the present invention, when converting into a circular nucleic acid template using conjugation, it may be characterized in that adhesive ends are generated at both ends by treatment with restriction enzymes, and then these are joined through ligase. In this case, as in one embodiment of the present invention, it may be characterized in that the ligation is performed without performing a DNA purification process. The "DNA purification process" refers to purifying and obtaining only the nucleic acid strand to be converted into a circular template by removing the cleavage fragment except for the desired nucleic acid strand after treatment with a restriction enzyme.

In one embodiment of the present invention, when converting into a circular nucleic acid template using infusion cloning, the same sequence must be located at both ends of the amplified nucleic acid strand, so G-tetramerization is also performed at the 5' end of the reverse primer. The sequence was added so that both ends of the amplified sequence were identical to the G-tetramerization sequence.

Accordingly, in the present invention, the primer pair may be characterized in that complementary nucleic acid sequences exist at the 5' end of the Forward primer and the 5' end of the Reverse primer in the reverse order.

In one embodiment of the present invention, after amplifying a linear nucleic acid strand including a promoter sequence, a target sequence, and a G-quadruplex sequence from a vector, ligation or infusion cloning Two methods were used to convert the original nucleic acid. Accordingly, in the present invention, step (b) may be characterized in that the circular nucleic acid template is converted through conjugation or infusion cloning, but is not limited thereto.

As used herein, "ligation" refers to linking two nucleic acid ends. Conjugation may be one in which both ends of one nucleic acid strand or the ends of different nucleic acid strands are connected, and in step (b) of the present invention, the 3' and 5 ends of the nucleic acid strand amplified for conversion into a circular nucleic acid template 'After the end is cut with the same restriction enzyme, it can be spliced.

In one embodiment of the present invention, when the amplified nucleic acid strand is converted into a cyclic nucleic acid template through ligation, it may be characterized by including the same restriction enzyme cleavage site at both ends of the amplified nucleic acid strand, , therefore, both ends of the amplified nucleic acid strand can be cleaved using the same restriction enzyme for conjugation. In the present invention, the restriction enzyme for cutting the amplified nucleic acid strand can be used without limitation, and depending on the restriction enzyme used, the cut end may be a sticky end or a blunt end, It is preferable that it is an adhesive end.

As used herein, the term "infusion cloning" refers to a Gibson assembly method using crossover of double-stranded nucleic acid strands having the same sequence. The "Gibson assembly method" is a DNA binding method using the crossover of double-stranded nucleic acid strands devised by Daniel G. Gibson, and detailed protocols and principles are described in Nature Methods. 6 (5): 343-345.

In one embodiment of the present invention, when converted into a circular nucleic acid template by the infusion cloning method, both ends of the amplified linear nucleic acid strand may include the same sequence. In an embodiment of the present invention, a primer designed to amplify a nucleic acid strand including a G-tetramerization sequence at both ends of the linear nucleic acid strand was amplified and then converted into a circular nucleic acid template by an infusion cloning method.

As used herein, the term “nucleic acid template” refers to a nucleic acid strand that can serve as a template for nucleic acid replication or transcription. In the present invention, the nucleic acid template may be, for example, a DNA or RNA template, preferably a DNA template, particularly a double-stranded nucleic acid template, but is not limited thereto. In the present invention, the amplified nucleic acid strand of step (b) includes a promoter sequence, a target sequence, and a G-tetramerization sequence, and, if necessary, without limitation 5'UTR, additional target sequences, such as sequences. can

As used herein, the term “circular nucleic acid template” refers to a circular nucleic acid strand serving as a template for transcription or replication. The circular nucleic acid template can be used as a template for amplification or transcription techniques called "Rolling Circle" or "Rolling Circle". In the present invention, it is used as a rolling circle amplification (RCA) and a rolling circle transcription (RCT). The RCA and RCT are selectively used depending on the type of nucleic acid to be prepared. RCA is used for the production of DNA complementary to the template, and RCT is used for the production of RNA complementary to the template. When RCT or RCA is performed using the circular nucleic acid template, the polymerization reaction does not end until the material (e.g., dNTP, ddNTP) is depleted, and a nucleic acid having an N fold from one template can be prepared. , characterized in that it exhibits a higher production yield than the linear template. In order to ensure that the transcription is continued rather than a single fold, the transcription termination sequence can optionally be removed. Accordingly, the nucleic acid template of the present invention may be characterized in that it does not include a transcription termination sequence.

In the present invention, the circular nucleic acid template may be a double-stranded nucleic acid, and in this case, the nucleic acid is synthesized using the antisense strand as a template.

In another aspect, the present invention relates to a nucleic acid template for producing a hydrogelled nucleic acid prepared by the method for producing the circular nucleic acid template for producing a hydrogelled nucleic acid.

In the present invention, the nucleic acid template for preparing the hydrogelled nucleic acid comprises a sequence complementary to a promoter; a sequence complementary to the target sequence; and a G-quadruplex motif sequence.

In the present invention, when the nucleic acid template for preparing the hydrogelled nucleic acid is double-stranded, a sequence complementary to the promoter; a sequence complementary to the target sequence; and a G-quadruplex motif sequence is included in the antisense strand, and a nucleic acid is synthesized using this as a template.

The nucleic acid template for preparing a hydrogelled nucleic acid of the present invention includes a sequence complementary to a promoter; a sequence complementary to the target sequence; and a G-quadruplex motif sequence. A nucleic acid (DNA or RNA, etc.) obtained by performing a rolling circle amplification (RCA) and a rolling circle transcription (RCT) using the nucleic acid template of the present invention as a template forms a G-quartet to form a hydro gelled.

Since hydrogelled nucleic acids have stronger physical properties than single-stranded ones, their stability can be remarkably increased, and can be usefully used as a platform technology for producing peptides or proteins therefrom.

The hydrogelled nucleic acid template of the present invention and a protein synthesis system using the same are disclosed in detail in Korean Patent Application No. 2019-0136416 of the present inventors.

As used herein, the term “linkage” means that nucleic acids or nucleic acid strands having a specific function (promoter, target sequence, G-tetramerization motif/sequence, etc.) are operably linked to each other, and the “operably linked” refers to each other Refers to two components placed in a functional relationship. The term "operably linked", when used in the context of a G-tetramerization motif, includes a sequence complementary to the target sequence when amplified or transcribed into a template from the G-tetramerization motif sequence linked to the target sequence. It means that the nucleic acid strand is capable of forming a G-tetramerized structure. Each linked sequence may be located upstream and/or downstream of each component. The linking may be achieved by ligation at the restriction site. If such a site does not exist, a synthetic oligonucleotide adapter or linker is used according to conventional practice. However, each element need not be contiguous to be operatively connected.

In another aspect, the present invention relates to a method for producing a hydrogelized nucleic acid comprising the step of amplifying or transcribed using the circular nucleic acid template prepared by the above production method as a template.

In the present invention, the hydrogelled nucleic acid may be characterized in that RNA or DNA.

In the present invention, the amplification refers to synthesizing a nucleic acid having the same sequence using a nucleic acid template as a template. The amplification of the present invention can be performed without limitation using a variety of methods. For example, to be performed through Rolling Circle RCA, Polymerase Chain Reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated amplification (TMA). In the present invention, since the circular nucleic acid template is used as a template, it is preferable to use RCA, but the present invention is not limited thereto. For the amplification, an appropriate nucleic acid polymerase (Polymerase), primer, etc. may be selected or manufactured by a person skilled in the art and used.

In the present invention, the transcription (transcription) is one of the DNA expression process. In the present invention, the transcription means synthesizing RNA using the nucleic acid template as a template. The transcription of the present invention may be performed without limitation using various methods, but it is preferable to perform a rolling circle transcription (RCT) method.

The salt conditions in the RNA solution in which the G-quadruplex structure of RNA is formed was confirmed through a specific flatness tendency according to the formation of the G-quadruplex structure of nucleic acids. Specifically, the formation of the G-quadruplex structure of the RNA through the RCT of the above Example in the conditions of 0, 0.1, 1, 10 or 100 mM KCl, NaCl and MgCl2 salt concentration was confirmed by circular dichroism analysis. As a result, under the conditions of 100 mM KCl and NaCl, a synthetic RNA having a G-quadruplex sequence of a half (containing only two sets of consecutive C (cytosine) sequences encoding continuous G, not all four sets) was produced. It formed a G-quadruplex structure and showed a tendency to flatten out from other conditions. In addition, it was found that the synthetic RNA having the mixed G-quadruplex sequence was not affected by the salt conditions, and thus it was distinguished from the synthetic RNA having the G-quadruplex sequence.

In the present invention, in order to promote the formation of a G- quadruplex of the produced nucleic acid to promote hydrogelation, the method may further include adding a cation, for example, most preferably potassium ion or sodium ion. .

In the present invention, when the target sequence included in the nucleic acid template has a sequence size of 150 bp or more or forms a secondary structure that inhibits hydrogelation of the produced nucleic acid, the productivity of the hydrogelled nucleic acid is lowered. In particular, since the target sequence encoding a high molecular weight protein has a very large sequence size, hydrogelation of the nucleic acid is very difficult.

Accordingly, the present inventors, in one embodiment, when adding a double-wide band template having a short sequence of 30 to 100 bp or less, the length of the target sequence of the nucleic acid template is 150 bp or more, or the hydrogelation of the produced nucleic acid is inhibited In the case of forming a secondary structure of In addition, in one embodiment of the present invention, when the circular template for producing hydrogelled nucleic acid of the present invention and the double-wide band template are amplified or transcribed together, it is confirmed that the difference in mechanical properties and strength changes according to the concentration, and the protein The concentration of the double-wide band template most suitable for expression was optimized.

Therefore, in the present invention, a second nucleic acid template comprising a sequence complementary to a promoter sequence, a second target sequence, and a G-quadruplex motif sequence is simultaneously amplified or transcribed. It may further include a step, wherein the second target sequence may be characterized as a short nucleotide sequence of less than 150 bp, preferably 30 to 120 bp, more preferably 60 to 90 bp in length.

In the present invention, the length of the second target sequence may have an effect on the formation of a double-wide band, and the sequence may have a relatively insignificant effect. In the present invention, the second target sequence may be a poly T sequence in which thymine is repeated.

In an embodiment of the present invention, a poly T sequence in which 30 to 120 thymines are repeated was used as the second target sequence, but the present invention is not limited thereto.

As used herein, the term "secondary structure that inhibits hydrogelation of nucleic acid" means a secondary structure of a nucleic acid formed by various characteristics such as the length of a base sequence and GC content, for example, a loop structure, GC There is a structure in which is included in an amount of 30% to 75%, but is not limited thereto.

As used herein, the term "second target sequence" refers to a sequence included in the second nucleic acid template (double-wide band template) and an inserted short nucleotide sequence that further improves hydrogelation of nucleic acids. In the present specification, when referring to a double-wide band template, the double-wide band template is a second nucleic acid template, and the circular nucleic acid template for producing hydrogelled nucleic acids of the present invention is a first nucleic acid template to distinguish it from a double-wide band template. is referred to as

In the present invention, the term "second nucleic acid template" is used interchangeably with the same meaning as "double-band template" in the present invention, and 'first' and 'second' are numbers assigned to distinguish the two templates. am. "Double-wide band" is a first nucleic acid template (a circular template for producing hydrogelled nucleic acids) and a G-quartet sequence of each nucleic acid produced using the double-wide band template as a template to form a G-quartet. It means the structure to be The double-wide band prevents the formation of a hydrogel from being inhibited because the distance between the G- quadruplexes is too far and prevents the formation of G- quadrules by the unintended secondary structure of the target sequence from being inhibited, so that it is more effective It can induce hydrogelation.

As confirmed in an embodiment of the present invention, when the hydrogelled nucleic acid is prepared using the double-wide band template together, it was confirmed that the mechanical properties change according to the concentration, and simply increase the ratio of the double-wide band template Accordingly, it was confirmed that hydrogelation of nucleic acids does not increase, but when it has a specific ratio, it shows hydrogelation appropriate for protein expression and affects protein expression efficiency.

In the present invention, the second nucleic acid template (double wide band template) may be mixed with the first nucleic acid template in a molar ratio of 1:1 to 600:1 and amplified or transcribed, preferably 75: Amplification or transcription may be characterized by mixing in a molar ratio of 1 to 300:1, more preferably in a molar ratio of 150:1 to 300:1.

In the present invention, it is preferable that the first nucleic acid template and the second nucleic acid template are mixed and amplified or transcribed at the same time. can

In the present invention, amplification may be performed using a nucleic acid amplification kit or method known in the art. For example, there are Rolling Circle RCA, Polymerase Chain Reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated amplification (TMA) methods. , it is preferable to use an isothermal amplification method, but is not limited thereto. In the present invention, it is preferable to amplify through a rolling circle amplification (Rolling Circle RCA).

In the present invention, transcription can be performed through a DNA transcription kit or method known in the art, and changes in RNA polymerase, dNTP, reagents, etc. can be easily made by those skilled in the art.

In another aspect, the present invention relates to a hydrogelled nucleic acid prepared by the method for preparing the hydrogelled nucleic acid.

In the present invention, the hydrogelled nucleic acid comprises a target sequence; And it is characterized in that it comprises a G- quadruplex sequence (G-quadruplex sequence) sequence.

Commercial protein production methods known to date have mainly been used to destroy the cytoplasm of living cells or chemically polymerize amino acids. However, these methods go through a very complicated production process in the cell disruption process and purification/separation process, and the produced protein is not normally expressed or is not well accepted in the body and causes an immune response, and the amino acid sequence in the polymerization of long-chain proteins It had disadvantages such as poor accuracy. Therefore, in recent years, the development of a cell-free protein synthesis system (cell-free or in vitro protein synthesis system) that does not use cells in the peptide or protein production process is being actively conducted.

The cell-free protein synthesis system uses key elements (ATP (adenosine triphosphate), amino acids, etc.) for protein synthesis by adding a substrate or enzyme to cell lysates or extracts. A method for synthesizing proteins in Such a cell-free protein synthesis system can overcome the disadvantages of the existing protein production method using cells, and cell-free protein synthesis uses only the intracellular machinery and its factors related to protein production in the cell. By artificially repeating only the protein synthesis process in a state in which the physiological control mechanism of the cell is excluded from extraction and mass production of the target protein in a short period of time, it is possible to synthesize the protein at high speed without going through the cell culture process as well as the cell membrane Compared to the cell culture process in which protein expression is carried out in the space of the cell wall and cell wall, it is a completely open system that does not have any physical barriers, and has the advantage of being able to freely modify the conditions for protein synthesis to apply it to various studies. In addition, the intracellular protein synthesis system has a great problem in its production and yield when the produced protein is cytotoxic. However, when a cell-free protein synthesis system is used, peptides or proteins can be freely produced in these problems. In addition, it is expected to increase productivity because it can be free from various external conditions such as pH, temperature, and ionic strength when protein is synthesized. However, despite having these advantages, it is not practically used because of the high cost of the reagents consumed due to the short reaction time and low protein production rate. In order to overcome these disadvantages, various attempts have been made to increase productivity by adding various chemicals such as iodoacetamide and glutathione or by developing a continuous flow system, but it is still insufficient for industrial use. the current situation.

In one embodiment of the present invention, when in vitro translation of the protein is performed by translating the hydrogelled nucleic acid transcribed from the nucleic acid template of the present invention as a template, protein expression compared to a negative control or linear DNA It was confirmed that this was improved by 4 to 6 times or more. In particular, it was confirmed that protein expression was further improved when using a nucleic acid template prepared using the infusion cloning method. In the case of producing a peptide or protein using the hydrogelled nucleic acid system by the method of the present invention, there is an advantage that the production yield can be maximized, and in particular, it can be usefully used in a cell-free synthesis system for synthesizing a peptide or protein.

Accordingly, in another aspect of the present invention, the present invention also includes the steps of (a') preparing a hydrogelled RNA by transcribed from the circular nucleic acid template for preparing the hydrogelled nucleic acid; and

It relates to a method for producing a peptide or protein comprising the step of translating the hydrogelled RNA and synthesizing the peptide or protein.

As used herein, "cell-free protein synthesis system (cell-free or in vitro protein synthesis system)" refers to a 'cell-free or in vitro synthesis system' or a 'cell-free peptide synthesis system' depending on the production target. (cell-free or in vitro peptide synthesis system)' or 'cell-free protein/peptide synthesis system' can be used interchangeably with the same meaning.

In one embodiment of the present invention, the hydrogel (hydrogel) RNA produced by the production method of the hydrogelized nucleic acid is translated to verify that the protein production efficiency is remarkably excellent even when using the cell-free production method It was confirmed that protein expression using the hydrogelled RNA generated by transcribed together with the double-wide band template of the present invention exhibits additional improvement in protein production at an appropriate concentration.

The method for producing a hydrogelled nucleic acid and hydrogelled nucleic acid of the present invention not only have very excellent structural stability outside the cell, but also have very excellent protein production capacity through a cell-free protein or peptide expression system, and thus the existing cell-free protein synthesis system Various limitations of this can be overcome, and thus it can be usefully used as a next-generation protein expression system.

In the present invention, the step (a') comprises a sequence complementary to the promoter sequence, a second target sequence having a length of 30bp to 120bp, and a G-quadruplex motif sequence. 2 After adding the nucleic acid template, it may be characterized in that it is transcribed to prepare a hydrogelled RNA.

In the present invention, the second nucleic acid template (double-wide band template) may be mixed with the first nucleic acid template in a molar ratio of 1:1 to 600:1 and amplified or transcribed, preferably 75: It may be characterized in that it is added and transferred in a molar ratio of 1 to 300:1, more preferably in a molar ratio of 150:1 to 300:1.

As used herein, the term “translation” refers to synthesizing a peptide or protein from RNA encoding a specific peptide or protein sequence through ribosomes.

In the present invention, in order to increase the surface area, it may be characterized in that the hydrogelled nucleic acid is pulverized and translated.

In the present invention, the method for producing a peptide or protein through translation of the protein may be characterized in that it is performed by an in vivo or cell-free protein/peptide synthesis system. Nucleic acid produced by using the template of the nucleic acid as a template has excellent stability outside the cell through hydrogelation, so it can be preferably used in a cell-free protein/peptide synthesis system.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Preparation of vector for preparing nucleic acid template for production of high molecular weight protein

A nucleic acid strand including a green fluorescent protein (GFP, green fluorescent protein) gene and a G-tetramerization sequence was cloned into pK7-vector, an E. coli expression vector (top of FIG. 1 ). Restriction enzymes used for cloning are NdeI and SalI. A nucleic acid strand including a green fluorescent protein (GFP) gene and a G-tetramerization sequence was synthesized by IDT (SEQ ID NOs: 3 and 4). Specifically, the GFP gene was amplified by PCR using the pK7-GFP-NdeI-F and pK7-GFP-SalI-R primers of [Table 1]. For PCR, after mixing the composition of Genetbio's HS Prime Taq DNA Polymerase product according to the manual, at 94°C for 10 minutes (1 cycle), at 94°C for 30 seconds, at 56°C for 30 seconds, at 72°C for 1 minute (30 cycles), 5 min (1 cycle) at 72°C. The pK7-vector and the green fluorescent protein gene were treated with the restriction enzymes NdeI and SalI of ThermoFisher, respectively, and both ends were cut (enzyme digestion) and then ligated using Promega's T4 DNA ligase product according to the manual. A vector (pK7-GFP) for preparing a nucleic acid template for GFP production was prepared.

Primer sequence for synthesis of synthesized nucleic acid strand and RCT template name order SEQ ID NO: GFP-G4
(sense strand) ATGAGCAAAG GTGAAGAACT GTTTACCGGC GTTGTGCCGA TTCTGGTGGA ACTGGATGGC GATGTGAACG GTCACAAATT CAGCGTGCGT GGTGAAGGTG AAGGCGATGC CACGATTGGC AAACTGACGC TGAAATTTAT CTGCACCACC GGCAAACTGC CGGTGCCGTG GCCGACGCTG GTGACCACCC TGACCTATGG CGTTCAGTGT TTTAGTCGCT ATCCGGATCA CATGAAACGT CACGATTTCT TTAAATCTGC AATGCCGGAA GGCTATGTGC AGGAACGTAC GATTAGCTTT AAAGATGATG GCAAATAA AACGCGCGCC GTTGTGAAAT TTGAAGGCGA TACCCTGGTG AACCGCATTG AACTGAAAGG CACGGATTTT AAAGAAGATG GCAATATCCT GGGCCATAAA CTGGAATACA ACTTTAATAG CCATAATGTT TATATTACGG CGGATAAACA GAAAAATGGC ATCAAAGCGA ATTTTACCGT TCGCCATAAC GTTGAAGATG GCAGTGTGCA GCTGGCAGAT CATTATCAGC AGAATACCCC GATTGGTGAT GGTCCGGTGC TGCTGCCGGA TAATCATTAT CTGAGCACGC AGACCGTTCT GTCTAAAGAT CCGAACGAGT TACG TGGGACCA CATGGTTCTG CACGAATAC AGT TACG TGGGACCA CATGGTTCTG CACGAATAC TAG TCGCCATAAC GTTGAAGATG GCAGTGTGCA 3 GFP-G4 (antisense strand) ACCCTAACCC TA TTATTTTT CGAACTGCGG ATGGCTCCAC GTAATACCTG CCGCATTCAC ATATTCGTGC AGAACCATGT GGTCCCGTTT TTCGTTCGGA TCTTTAGACA GAACGGTCTG CGTGCTCAGA TAATGATTAT CCGGCAGCAG CACCGGACCA TCCCAGCTAATCG GGGTATTC TG CACCGGACCA
GCACACTGCC ATCTTCAACG TTATGGCGAA CGGTAAAATT CGCTTTGATG CCATTTTTCT GTTTATCCGC CGTAATATAA ACATTATGGC TATTAAAGTT GTATTCCAGT TTATGGCCCA GGATATTGCC ATCTTCTTTA GTAAATCCGTGC CTTTCAGTCA TTCA ATGAACGGTCC ACGG
GCGCGCGTTT TATTTGCCAT CATCTTTAAA GCTAATCGTA CGTTCCTGCA CATAGCCTTC CGGCATTGCA GATTTAAAGA AATCGTGACG TTTCATGTGA TCCGGATAGC GACTAAAACA CTGAACGCCA TAGGTCAGGG TGGTTTCACCAG CGTCGGCCAC GGCACCGGCA GTTTGCCGGT
TCAGTTTGCC AATCGTGGCA TCGCCTTCAC CTTCACCACG CACGCTGAAT TTGTGACCGT TCACATCGCC ATCCAGTTCC ACCAGAATCG GCACAACGCC GGTAAACAGT TCTTCACCTT TGCTCAT 4 pK7-GFP-NdeI-F ATA CAT ATG AGC AAA GGT GAA GAA CTG 5 pK7-GFP-SalI-R CCG GTC GAC TTA TTT TTC GAA CTG CGG ATG G 6 pK7-T7-F2 ATA GGA TCC TAA TAC GAC TCA CTA TAG GGA GAC C 7 pK7-T7-R2 TAT GGA TCC ACC CTA ACC CTA TTA TTT TTC GAA CTG CGG ATG G 8

* In Table 1, boldface indicates a G-quartetization sequence (sense strand) or a G-quartet motif sequence (antisense strand). * In Table 1, and lines indicate restriction enzyme cleavage sites

Example 2: Preparation of circular nucleic acid template (ligation method)

Using the vector prepared in Example 1 as a template, using the pK-T7-F2 and pK-T7-R2 primers of [Table 1], PCR was performed to include the T7 promoter, 5' UTR, GFP gene and G-tetramerization sequence. By amplification, linear nucleic acid strands (T7-GFP-G4-BamHI, SEQ ID NOs: 9 and 10) were prepared. For PCR, after mixing the composition of Genetbio's HS Prime Taq DNA Polymerase product according to the manual, at 94°C for 10 minutes (1 cycle), at 94°C for 30 seconds, at 56°C for 30 seconds, at 72°C for 1 minute (30 cycles), 5 min (1 cycle) at 72°C. The prepared linear nucleic acid strand (T7-GFP-G4-BamHI) was treated at 37 °C for 30 minutes using ThermoFisher's BamHI to cut both ends, and then spliced for 3 hours at room temperature using Promega's T4 DNA ligase product. It was converted into a circular nucleic acid template through ligation (FIG. 2a).

amplified nucleic acid strand sequence name order SEQ ID NO: BamHI-T7-GFP-G4-BamHI
(sense strand) ATA GGATCC T AATACGACTC ACTATAGGGA GACCACAACG GTTTCCCTCT AGAAATAATT TTGTTTAACT TTAAGAAGGA GATATACATA TGAGCAAAGG TGAAGAACTG TTTACCGGCG
TTGTGCCGAT TCTGGTGGAA CTGGATGGCG ATGTGAACGG TCACAAATTC AGCGTGCGTG GTGAAGGTGA AGGCGATGCC ACGATTGGCA AACTGACGCT GAAATTTATC TGCACCACCG
GCAAACTGCC GGTGCCGTGG CCGACGCTGG TGACCACCCT GACCTATGGC GTTCAGTGTT TTAGTCGCTA TCCGGATCAC ATGAAACGTC ACGATTTCTT TAAATCTGCA ATGCCGGAAG
GCTATGTGCA GGAACGTACG ATTAGCTTTA AAGATGATGG CAAATATAAA ACGCGCGCCG TTGTGAAATT TGAAGGCGAT ACCCTGGTGA ACCGCATTGA ACTGAAAGGC ACGGATTTTA
AAGAAGATGG CAATATCCTG GGCCATAAAC TGGAATACAA CTTTAATAGC CATAATGTTT ATATTACGGC GGATAAACAG AAAAATGGCA TCAAAGCGAA TTTTACCGTT CGCCATAACG
TTGAAGATGG CAGTGTGCAG CTGGCAGATC ATTATCAGCA GAATACCCCG ATTGGTGATG GTCCGGTGCT GCTGCCGGAT AATCATTATC TGAGCACGCA GACCGTTCTG TCTAAAGATC
CGAACGAAAA ACGGGACCAC ATGGTTCTGC ACGAATATGT GAATGCGGCA GGTATTACGT GGAGCCATCC GCAGTTCGAA AAATAA TAGG GTTAGGGT GG ATCC ATA 9 T7-GFP-G4-BamHI (antisense strand) TAT GGATCC A CCCTAACCCT A TTATTTTTC GAACTGCGGA TGGCTCCACG TAATACCTGC CGCATTCACA TATTCGTGCA GAACCATGTG GTCCCGTTTT TCGTTCGGAT CTTTAGACAG AACGGTCTGC GTGCTCAGAT AATGATTATC CGGCAGCAGC ACCGG CAT
CTGCCAGCTG CACACTGCCA TCTTCAACGT TATGGCGAAC GGTAAAATTC GCTTTGATGC CATTTTTCTG TTTATCCGCC GTAATATAAA CATTATGGCT ATTAAAGTTG TATTCCAGTT TATGGCCCAG GATATTGCCA TCTTCTTTAA AATCCGTGCTGC TTATCATTCA ATGG
TTCACAACGG CGCGCGTTTT ATATTTGCCA TCATCTTTAA AGCTAATCGT ACGTTCCTGC ACATAGCCTT CCGGCATTGC AGATTTAAAG AAATCGTGAC GTTTCATGTG ATCCGGATAG CGACTAAAAC ACTGAACGCC ATAGGTCAGG GTGGTCACCA GCACCGCAGGCCA CGGG
AAATTTCAGC GTCAGTTTGC CAATCGTGGC ATCGCCTTCA CCTTCACCAC GCACGCTGAA TTTGTGACCG TTCACATCGC CATCCAGTTC CACCAGAATC GGCACAACGC CGGTAAACAG TTCTTCACCT TTGCTCATAT GTATATC TGTCC TTCTTAAAGT TACTAACAGA GGGAAGT TACTAACCAAAT TATT
CTATAGTGAG TCGTATTA GG ATCC TAT 10

* Bold text in Table 2 indicates a G-quadrued sequence (sense strand) or a G-quartet motif sequence (antisense strand).

* In Table 2, and lines indicate restriction enzyme (BamHI) cleavage sites

Example 3: Confirmation of whether DNA purification process is required when preparing nucleic acid template

After BamHI treatment, for ligation, it was confirmed that the circular nucleic acid template was generated according to the presence or absence of a DNA purification process (FIG. 2b). Purification of DNA means that, after treatment with BamHI, the cleaved fragment is removed, and only the nucleic acid strand for conversion into the original nucleic acid template is purified. In order to clearly confirm the conversion to the original nucleic acid template, the result of treatment with NEB's T7 exonuclease at room temperature for 30 minutes was confirmed. As a result, it was confirmed by 1% agarose gel electrophoresis that a circular nucleic acid template was generated only from a gene for which DNA purification was not performed (FIG. 2c).

To verify that T7 exonuclease specifically degrades only the nucleic acid template, the linear nucleic acid strand and the circular nucleic acid template were treated with T7 exonuclease at room temperature for 10 minutes, 20 minutes, and 30 minutes, respectively. All of the linear nucleic acid strands were degraded from 10 minutes ( FIG. 3D ), and the circular DNA showed the same state as the control group (not treated with T7 exonuclease) in all treatment groups ( FIG. 3E ). This means that the T7 exonuclease degrades only the linear nucleic acid strand.

Example 4: Preparation of Hydrogelled Nucleic Acid Using the Prepared Nucleic Acid Template

In order to confirm the correlation between the morphological difference between the transcript of the circular nucleic acid template and the linear nucleic acid strand prepared in Example 2 and the amount of RNA obtained by concentration of the nucleic acid template, New England Biolabs' HiScribe?? In vitro transcription was performed using the T7 High Yield RNA Synthesis Kit product according to the manual. In the result of using a linear nucleic acid strand as a template, a monomer-sized transcript of the GFP gene was generated (Fig. 4f, lanes 1, 3, 5), whereas in the result using a circular nucleic acid template as a template, a smeared band circular circulation was generated. It was confirmed that a large amount of transcript was produced by transcription (RCT) (FIG. 4f, lanes 2, 4, 6). The concentration of the nucleic acid template and the yield of the transcript were proportional, but not directly proportional (FIG. 4g), and showed the most efficient transcript (RNA) obtaining yield at 100 ng to 200 ng.

Example 5: Preparation of Nucleic Acid Template (Infusion Cloning Method) and Transcript (Identification of Hydrogelled Nucleic Acid)

The vector prepared in Example 1 was amplified by PCR using the primers pK7-GFP-infusion-F3 and pK7-GFP-infusion-R4 of Table 3 as a template to prepare a linear nucleic acid strand (G4-T7-GFP-G4). (FIG. 5A, SEQ ID NOs: 11 and 12). For PCR, after mixing the composition of Genetbio's HS Primer Taq DNA Polymerase product according to the manual, at 94°C for 10 minutes (1 cycle), at 94°C for 30 seconds, at 56°C for 30 seconds, at 72°C for 1 minute (30 cycles), 5 min (1 cycle) at 72°C. The produced linear nucleic acid strand (G4-T7-GFP-G4) was converted into a circular template by processing at 50 °C for 15 minutes using Takara's In-Fusion® HD Cloning kit (Fig. 5b).

Sequences of amplified nucleic acid strands and primers for a method for preparing a circular nucleic acid template using infusion cloning name order SEQ ID NO: G4-T7-GFP-G4
(sense strand) TAA TAGGGTT AGGGT TAATA CGACTCACTA TAGGGAGACC ACAACGGTTT CCCTCTAGAA ATAATTTTGT TTAACTTTAA GAAGGAGATA TACATATGAG CAAAGGTGAA GAACTGTTTA
CCGGCGTTGT GCCGATTCTG GTGGAACTGG ATGGCGATGT GAACGGTCAC AAATTCAGCG TGCGTGGTGA AGGTGAAGGC GATGCCACGA TTGGCAAACT GACGCTGAAA TTTATCTGCA
CCACCGGCAA ACTGCCGGTG CCGTGGCCGA CGCTGGTGAC CACCCTGACC TATGGCGTTC AGTGTTTTAG TCGCTATCCG GATCACATGA AACGTCACGA TTTCTTTAAA TCTGCAATGC
CGGAAGGCTA TGTGCAGGAA CGTACGATTA GCTTTAAAGA TGATGGCAAA TATAAAACGC GCGCCGTTGT GAAATTTGAA GGCGATACCC TGGTGAACCG CATTGAACTG AAAGGCACGG
ATTTTAAAGA AGATGGCAAT ATCCTGGGCC ATAAACTGGA ATACAACTTT AATAGCCATA ATGTTTATAT TACGGCGGAT AAACAGAAAA ATGGCATCAA AGCGAATTTT ACCGTTCGCC
ATAACGTTGA AGATGGCAGT GTGCAGCTGG CAGATCATTA TCAGCAGAAT ACCCCGATTG GTGATGGTCC GGTGCTGCTG CCGGATAATC ATTATCTGAG CACGCAGACC GTTCTGTCTA AAGATCCGAA CGAAAAACGG GACCACATGG TTCTGCACGA GACCACATGG TTCTGCACGA TAG TAG AA TAACGGTAGG 11 G4-T7-GFP-G4 (antisense strand) ACCCTAACCC TA TTA TTTTT CGAACTGCGG ATGGCTCCAC GTAATACCTG CCGCATTCAC ATATTCGTGC AGAACCATGT GGTCCCGTTT TTCGTTCGGA TCTTTAGACA GAACGGTCTG CGTGCTCAGA TAATGATTAT CCGGCAGCAG CACCGGACCA TCCCAGCTAATCG GGGTATTC TG
GCACACTGCC ATCTTCAACG TTATGGCGAA CGGTAAAATT CGCTTTGATG CCATTTTTCT GTTTATCCGC CGTAATATAA ACATTATGGC TATTAAAGTT GTATTCCAGT TTATGGCCCA GGATATTGCC ATCTTCTTTA GTAAATCCGTGC CTTTCAGTCA TTCA ATGAACGGTCC ACGG
GCGCGCGTTT TATATTTGCC ATCATCTTTA AAGCTAATCG TACGTTCCTG CACATAGCCT TCCGGCATTG CAGATTTAAA GAAATCGTGA CGTTTCATGT GATCCGGATA GCGACTAAAA CACTGAACGC CATAGGTCAG GGTGGTTTCACC AGCGTCGGCC ACGGCACCGG CACATTTGCCG GTGG
CGTCAGTTTG CCAATCGTGG CATCGCCTTC ACCTTCACCA CGCACGCTGA ATTTGTGACC GTTCACATCG CCATCCAGTT CCACCAGAAT CGGCACAACG CCGGTAAACA GTTCTTCACC TTTGCTCATA TGTATATCTC CTTCTTAACTATAGAACAAAA TTATTTCTAG AGGGAAACCG CGTCAGTTTG
GTCGTATTA A CCCTAACCCT A TTA 12 pK7-GFP-infusion-F3 TAATAGGGTT AGGGT TAATA CGACTCACTA TAGGGAGACCA 13 pK7-GFP-infusion-R3 ACCCTAACCC TATTA TTTTT CGAACTGCGG ATGGC 14

* Underline indicates some sequences of primers designed to have the same sequence at both ends for infusion cloning.

* Bold text indicates a G-quadrelization sequence (sense strand) or a G-quartet motif sequence (antisense strand).

As a result of treating NEB's T7 exonuclease at room temperature for 30 minutes at room temperature to check whether the conversion to the original nucleic acid template was performed, all the linear nucleic acid strands resulting from PCR were degraded, whereas the nucleic acid template subjected to infusion cloning was not degraded, so it was converted to a circular nucleic acid template. It was confirmed that it was converted (FIG. 5c). HiScribe from New England Biolabs?? As a result of in vitro transcription using the T7 High Yield RNA Synthesis Kit, it was confirmed that the RCT transcript was produced by the circular circulation transcription technique in the smeared band form from the donut as a template (FIG. 5d).

Example 6: Verification of GFP expression efficiency using a circular nucleic acid template

In vitro translation was performed using the hydrogelled nucleic acids produced in Examples 2 and 4 above. NEBExpress® Cell-free E. coli Protein from New England Biolabs, using 2.5 μg of linear nucleic acid strand, circular nucleic acid template (conjugation method, donut 1), and RNA transcribed from circular nucleic acid template (infusion cloning method, donut 2). In vitro protein expression was performed using the Synthesis System, and additionally, RNA gel adjusted to a final concentration of 1% by adding 2% agarose gel according to the amount of each RNA added was added for 16 hours at 37°C. Protein expression was carried out. As a result of measuring the fluorescence of the expressed green fluorescent protein, it was confirmed that the RFU (relative fluorescence units) value of the control linear nucleic acid strand was 5621.9, whereas donut1: 22,000, donut2: 30000, indicating about 4 to 6 times higher RFU value. did Relatively, donut1 + agarose added with agarose gel: 16,000, donut2 + agarose: 21,000 showed a lower RFU value than the condition without adding agarose gel (FIG. 6a). As a result of confirming 4 μg of green fluorescent protein expressed by each condition by 12% SDS-PAGE, a band of 26.86 kDa green fluorescent protein was observed (FIG. 6b). As a result, it means that the circular nucleic acid template of the present invention normally expressed GFP and showed higher expression efficiency than the linear nucleic acid strand.

Example 7: RNA hydrogel (hydrogel) formation and physical properties confirmation using a double-wide band template

T7 promoter (the T7 promoter is separated into two parts and is present at 5' and 3' of the oligonucleotide), 30 poly T, 60 poly T, 90 poly T, 120 poly T and an oligonucleotide containing a G-tetramerization motif The T7 promoter sequence is nicked by mixing an intact oligonucleotide complementary to the entire sequence of the isolated promoter to 45 μM and annealing from 95°C to 4°C in 100 mM NaCl condition. to form a double-stranded The annealed T7 promoter nick was ligated using Promega's T4 DNA ligase product at a concentration of 10 μM according to the manual, and the nick was removed to include the T7 promoter-each Poly T sequence-G-tetramerization motif. A circular double-wide band template (SEQ ID NOs: 15 to 18) was prepared.

double wide band template sequence name sequence (5' -> 3') SEQ ID NO: double wide band template
: G4-30T (2nd template) ATAGTGAGTC GTATTA ACCC TAACCCTA TT TTTTTTTTTT TTTTTTTTTT TTTTTTTT AT CCCT 15 Double wide band template: G4-60T (2nd template) ATAGTGAGTC GTATTA ACCC TAACCCTA TT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTT AT CCCT 16 Double wide band template: G4-90T (2nd template) ATAGTGAGTC GTATTA ACCC TAACCCTA TT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTT TTTTTTTT AT CCCT 17 Double wide band template: G4-120T (2nd template) ATAGTGAGTC GTATTA ACCC TAACCCTA TT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTT AT CCCT 18

In vitro transcription was performed by adding double-wide band templates (G4-30T, -60T, -90T, -120T) at various concentrations together with the circular nucleic acid template of Example 2 obtained by the conjugation method. Based on protein expression, 1 μM of double-wide band template (G4-30T, -60T, -90T, -120T) was set as a negative control group, and 20 ng of a circular nucleic acid template for GFP expression was set as a positive control group. For the experimental group, transcription was performed by adding 0.125 μM to 1 μM of a double-wide band template to 20 ng of a circular nucleic acid template for GFP expression. As a result, it was evaluated whether the RNA hydrogel was formed and the mechanical properties changed according to the concentration of the double-wide band template. When the mechanical properties of the negative control group were set to the highest (+++++), it was confirmed that the appropriate strength was exhibited according to the size and concentration of the double-wide band template (Table 5).

Composition of each control group and experimental group, and whether or not hydrogel was formed and strength Number Name Template type template concentration Wideband type Gelation One negative - - 30T-1 μM +++++ 2 Positive Donut 20 ng - - 3 Angel Donut 20 ng 30T-0.125 μM - 4 Angel Donut 20 ng 30T-0.25 μM - 5 Angel Donut 20 ng 30T-0.5 μM ++ 6 Angel Donut 20 ng 30T-0.75 μM +++++ 7 Angel Donut 20 ng 30T-1 μM +++++ 8 negative - - 60T-1 μM +++++ 9 Positive Donut 20 ng - - 10 Angel Donut 20 ng 60T-0.125 μM + 11 Angel Donut 20 ng 60T-0.25 μM +++ 12 Angel Donut 20 ng 60T-0.5 μM +++++ 13 Angel Donut 20 ng 60T-0.75 μM +++++ 14 Angel Donut 20 ng 60T-1 μM +++++ 15 negative - - 90T-1 μM +++++ 16 Positive Donut 20 ng - - 17 Angel Donut 20 ng 90T-0.125 μM + 18 Angel Donut 20 ng 90T-0.25 μM +++++ 19 Angel Donut 20 ng 90T-0.5 μM +++++ 20 Angel Donut 20 ng 90T-0.75 μM +++++ 21 Angel Donut 20 ng 90T-1 μM +++++ 22 negative - - 120T-1 μM +++++ 23 Positive Donut 20 ng - - 24 Angel Donut 20 ng 120T-0.125 μM + 25 Angel Donut 20 ng 120T-0.25 μM +++++ 26 Angel Donut 20 ng 120T-0.5 μM +++++ 27 Angel Donut 20 ng 120T-0.75 μM +++++ 28 Angel Donut 20 ng 120T-1 μM ++++

Example 8: Verification of GFP expression efficiency when using a double-wide band template

In vitro translation was performed using 28 RNA hydrogels prepared in Example 7 to express green fluorescent protein. A total of 28 experimental groups including negative control, positive control, and GFP angel were subjected to protein expression at 37°C for 4 hours using New England Biolabs' NEBExpress® Cell-free E. coli Protein Synthesis System, and the expressed green fluorescent protein was RFU values were measured (Table 6).

RFU value of green fluorescent protein in each control group and experimental group Number Name Wideband type RFU Number Name Wideband type RFU One negative 30T-
1 μM 168.46 15 negative 90T-
1 μM 168.46 2 Positive - 4312.26 16 Positive - 4312.26 3 Angel 30T-
0.125 μM 4614.99 17 Angel 90T-
0.125 μM 7075.71 4 Angel 30T-0.25 μM 4561.78 18 Angel 90T-
0.25 μM 7406.6 5 Angel 30T-0.5 μM 7257.58 19 Angel 90T-
0.5 μM 8556.68 6 Angel 30T-0.75 μM 7007.31 20 Angel 90T-
0.75 μM 9388.27 7 Angel 30T-
1 μM 7185.96 21 Angel 90T-
1 μM 9658.04 8 negative 60T-
1 μM 168.46 22 negative 120T-
1 μM 168.46 9 Positive - 4312.26 23 Positive - 4312.26 10 Angel 60T-
0.125 μM 6320.14 24 Angel 120T-
0.125 μM 4486.84 11 Angel 60T-0.25 μM 6950.44 25 Angel 120T-
0.25 μM 6813.15 12 Angel 60T-0.5 μM 6132.26 26 Angel 120T-
0.5 μM 6666.26 13 Angel 60T-0.75 μM 7492.49 27 Angel 120T-
0.75 μM 8943.02 14 Angel 60T-
1 μM 9491.85 28 Angel 120T-
1 μM 8098.43

As a result, when the double-wide band template was added at a higher concentration than the negative control group (168.46) and the positive control group (4312.26), the RFU value showed a tendency to increase. In particular, GFP angel #14 (60T-1 μM) is 9491.85, GFP angel #20 (90T-0.75 μM) is 9388.27, GFP angel #21 (90T-1 μM) is 9658.04, GFP angel #27 (120T-0.75 μM) is was 8943.02, which showed 120%, 118%, 124%, and 107% higher RFU values compared to the positive control, respectively, and observed a protein expression synergistic effect due to the double-wide band (FIG. 7).

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby It will be obvious. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

<110> Progeneer Inc. <120> Manufacturing method of circular nucleic acid template for producing high molecular weight protein using hydrogelated nucleic acid <130> P20-B142 <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> G-quadruplex sequence <400> 1 tagggttagg gt 12 <210> 2 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> G-quadruplex motif sequence <400> 2 accctaaccc ta 12 <210> 3 <211> 727 <212> DNA <213> Artificial Sequence <220> <223> GFP-G4_Sense <400> 3 atgagcaaag gtgaagaact gtttaccggc gttgtgccga ttctggtgga actggatggc 60 gatgtgaacg gtcacaaatt cagcgtgcgt ggtgaaggtg aaggcgatgc cacgattggc 120 aaactgacgc tgaaatttat ctgcaccacc ggcaaactgc cggtgccgtg gccgacgctg 180 gtgaccaccc tgacctatgg cgttcagtgt tttagtcgct atccggatca catgaaacgt 240 cacgatttct ttaaatctgc aatgccggaa ggctatgtgc aggaacgtac gattagcttt 300 aaagatgatg gcaaataaaa cgcgcgccgt tgtgaaattt gaaggcgata ccctggtgaa 360 ccgcattgaa ctgaaaggca cggattttaa agaagatggc aatatcctgg gccataaact 420 ggaatacaac tttaatagcc ataatgttta tattacggcg gataaacaga aaaatggcat 480 caaagcgaat tttaccgttc gccataacgt tgaagatggc agtgtgcagc tggcagatca 540 ttatcagcag aataccccga ttggtgatgg tccggtgctg ctgccggata atcattatct 600 gagcaccgcag accgttctgt ctaaagatcc gaacgaaaaa cgggaccaca tggttctgca 660 cgaatatgtg aatgcggcag gtattacgtg gagccatccg cagttcgaaa aataataggg 720 taggggt 727 <210> 4 <211> 727 <212> DNA <213> Artificial Sequence <220> <223> GFP-G4_AntiSense <400> 4 accctaaccc tattattttt cgaactgcgg atggctccac gtaatacctg ccgcattcac 60 atattcgtgc agaaccatgt ggtcccgttt ttcgttcgga tctttagaca gaacggtctg 120 cgtgctcaga taatgattat ccggcagcag caccggacca tcaccaatcg gggtattctg 180 ctgataatga tctgccagct gcacactgcc atcttcaacg ttatggcgaa cggtaaaatt 240 cgctttgatg ccatttttct gtttatccgc cgtaatataa acattatggc tattaaagtt 300 gtattccagt ttatggccca ggatattgcc atcttcttta aaatccgtgc ctttcagttc 360 aatgcggttc accagggtat cgccttcaaa tttcacaacg gcgcgcgttt tatttgccat 420 catctttaaa gctaatcgta cgttcctgca catagccttc cggcattgca gatttaaaga 480 aatcgtgacg tttcatgtga tccggatagc gactaaaaca ctgaacgcca taggtcaggg 540 tggtcaccag cgtcggccac ggcaccggca gtttgccggt ggtgcagata aatttcagcg 600 tcagtttgcc aatcgtggca tcgccttcac cttcaccacg cacgctgaat ttgtgaccgt 660 tcacatcgcc atccagttcc accagaatcg gcacaacgcc ggtaaacagt tcttcacctt 720 tgctcat 727 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pK7-GFP-NdeI-Forward Primer <400> 5 atacatatga gcaaaggtga agaactg 27 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> pK7-GFP-SalI-Reverse Primer <400> 6 ccggtcgact tatttttcga actgcggatg g 31 <210> 7 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> pK7-T7-F2 (Forward Primer) <400> 7 ataggatcct aatacgactc actataggga gacc 34 <210> 8 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> pK7-T7-R2 (Reverse Primer) <400> 8 tatggatcca ccctaaccct attatttttc gaactgcgga tgg 43 <210> 9 <211> 827 <212> DNA <213> Artificial Sequence <220> <223> BamHI-T7-GFP-G4-BamHI_Sense <400> 9 ataggatcct aatacgactc actataggga gaccacaacg gtttccctct agaaataatt 60 ttgtttaact ttaagaagga gatatacata tgagcaaagg tgaagaactg tttaccggcg 120 ttgtgccgat tctggtggaa ctggatggcg atgtgaacgg tcacaaattc agcgtgcgtg 180 gtgaaggtga aggcgatgcc acgattggca aactgacgct gaaatttatc tgcaccaccg 240 gcaaactgcc ggtgccgtgg ccgacgctgg tgaccaccct gacctatggc gttcagtgtt 300 ttagtcgcta tccggatcac atgaaacgtc acgatttctt taaatctgca atgccggaag 360 gctatgtgca ggaacgtacg attagcttta aagatgatgg caaatataaa acgcgcgccg 420 ttgtgaaatt tgaaggcgat accctggtga accgcattga actgaaaggc acggatttta 480 aagaagatgg caatatcctg ggccataaac tggaatacaa ctttaatagc cataatgttt 540 atattacggc ggataaacag aaaaatggca tcaaagcgaa ttttaccgtt cgccataacg 600 ttgaagatgg cagtgtgcag ctggcagatc attatcagca gaataccccg attggtgatg 660 gtccggtgct gctgccggat aatcattatc tgagcacgca gaccgttctg tctaaagatc 720 cgaacgaaaa acgggaccac atggttctgc acgaatatgt gaatgcggca ggtattacgt 780 ggagccatcc gcagttcgaa aaataatagg gttagggtgg atccata 827 <210> 10 <211> 827 <212> DNA <213> Artificial Sequence <220> <223> BamHI-T7-GFP-G4-BamHI_AntiSense <400> 10 tatggatcca ccctaaccct attatttttc gaactgcgga tggctccacg taatacctgc 60 cgcattcaca tattcgtgca gaaccatgtg gtcccgtttt tcgttcggat ctttagacag 120 aacggtctgc gtgctcagat aatgattatc cggcagcagc accggaccat caccaatcgg 180 ggtattctgc tgataatgat ctgccagctg cacactgcca tcttcaacgt tatggcgaac 240 ggtaaaattc gctttgatgc catttttctg tttatccgcc gtaatataaa cattatggct 300 attaaagttg tattccagtt tatggcccag gatattgcca tcttctttaa aatccgtgcc 360 tttcagttca atgcggttca ccagggtatc gccttcaaat ttcacaacgg cgcgcgtttt 420 atatttgcca tcatctttaa agctaatcgt acgttcctgc acatagcctt ccggcattgc 480 agatttaaag aaatcgtgac gtttcatgtg atccggatag cgactaaaac actgaacgcc 540 ataggtcagg gtggtcacca gcgtcggcca cggcaccggc agtttgccgg tggtgcagat 600 aaatttcagc gtcagtttgc caatcgtggc atcgccttca ccttcaccac gcacgctgaa 660 tttgtgaccg ttcacatcgc catccagttc caccagaatc ggcacaacgc cggtaaacag 720 ttcttcacct ttgctcatat gtatatctcc ttcttaaagt taaacaaaat tatttctaga 780 gggaaaccgt tgtggtctcc ctatagtgag tcgtattagg atcctat 827 <210> 11 <211> 824 <212> DNA <213> Artificial Sequence <220> <223> G4-T7-GFP-G4_Sense <400> 11 taatagggtt agggttaata cgactcacta tagggagacc acaacggttt ccctctagaa 60 ataattttgt ttaactttaa gaaggagata tacatatgag caaaggtgaa gaactgttta 120 ccggcgttgt gccgattctg gtggaactgg atggcgatgt gaacggtcac aaattcagcg 180 tgcgtggtga aggtgaaggc gatgccacga ttggcaaact gacgctgaaa tttatctgca 240 ccaccggcaa actgccggtg ccgtggccga cgctggtgac caccctgacc tatggcgttc 300 agtgttttag tcgctatccg gatcacatga aacgtcacga tttctttaaa tctgcaatgc 360 cggaaggcta tgtgcaggaa cgtacgatta gctttaaaga tgatggcaaa tataaaacgc 420 gcgccgttgt gaaatttgaa ggcgataccc tggtgaaccg cattgaactg aaaggcacgg 480 attttaaaga agatggcaat atcctgggcc ataaactgga atacaacttt aatagccata 540 atgtttatat tacggcggat aaacagaaaa atggcatcaa agcgaatttt accgttcgcc 600 ataacgttga agatggcagt gtgcagctgg cagatcatta tcagcagaat accccgattg 660 gtgatggtcc ggtgctgctg ccggataatc attatctgag cacgcagacc gttctgtcta 720 aagatccgaa cgaaaaacgg gaccacatgg ttctgcacga atatgtgaat gcggcaggta 780 ttacgtggag ccatccgcag ttcgaaaaat aatagggtta gggt 824 <210> 12 <211> 824 <212> DNA <213> Artificial Sequence <220> <223> G4-T7-GFP-G4_AntiSense <400> 12 accctaaccc tattattttt cgaactgcgg atggctccac gtaatacctg ccgcattcac 60 atattcgtgc agaaccatgt ggtcccgttt ttcgttcgga tctttagaca gaacggtctg 120 cgtgctcaga taatgattat ccggcagcag caccggacca tcaccaatcg gggtattctg 180 ctgataatga tctgccagct gcacactgcc atcttcaacg ttatggcgaa cggtaaaatt 240 cgctttgatg ccatttttct gtttatccgc cgtaatataa acattatggc tattaaagtt 300 gtattccagt ttatggccca ggatattgcc atcttcttta aaatccgtgc ctttcagttc 360 aatgcggttc accagggtat cgccttcaaa tttcacaacg gcgcgcgttt tatatttgcc 420 atcatcttta aagctaatcg tacgttcctg cacatagcct tccggcattg cagatttaaa 480 gaaatcgtga cgtttcatgt gatccggata gcgactaaaa cactgaacgc cataggtcag 540 ggtggtcacc agcgtcggcc acggcaccgg cagtttgccg gtggtgcaga taaatttcag 600 cgtcagtttg ccaatcgtgg catcgccttc accttcacca cgcacgctga atttgtgacc 660 gttcacatcg ccatccagtt ccaccagaat cggcacaacg ccggtaaaca gttcttcacc 720 tttgctcata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag agggaaaccg 780 ttgtggtctc cctatagtga gtcgtatta ccctaaccct atta 824 <210> 13 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> pK7-GFP-infusion-F3 (Forward Primer) <400> 13 taatagggtt agggttaata cgactcacta tagggagacc a 41 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> pK7-GFP-infusion-R3 (Reverse Primer) <400> 14 accctaaccc tattattttt cgaactgcgg atggc 35 <210> 15 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Doublewide Band Template_30T <400> 15 atagtgagtc gtattaaccc taaccctatt tttttttttt tttttttttt ttttttttat 60 ccct 64 <210> 16 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> Doublewide Band Template_60T <400> 16 atagtgagtc gtattaaccc taaccctatt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt ttttttttat ccct 94 <210> 17 <211> 124 <212> DNA <213> Artificial Sequence <220> <223> Doublewide Band Template_90T <400> 17 atagtgagtc gtattaaccc taaccctatt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttat 120 ccct 124 <210> 18 <211> 154 <212> DNA <213> Artificial Sequence <220> <223> Doublewide Band Template_120T <400> 18 atagtgagtc gtattaaccc taaccctatt tttttttttt tttttttttt tttttttttt 60 ttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt ttttttttat ccct 154

Claims (16)

(a) a promoter sequence; target sequence; and amplifying a nucleic acid strand comprising a promoter sequence, a target sequence, and a G-quadruplex sequence in a vector comprising a G-quadruplex seqeuence sequence; and
(b) converting the amplified nucleic acid strand into a circular nucleic acid template to obtain a circular nucleic acid template for producing hydrogelled nucleic acid.
The method according to claim 1, wherein in step (b), the amplified nucleic acid strand is converted into a circular nucleic acid template through ligation or infusion cloning.
The method according to claim 1, wherein the circular nucleic acid template for preparing the hydrogelled nucleic acid is a DNA or RNA template.
The method according to claim 1, wherein the vector of step (a) is selected from the group consisting of pK7, pIVEX, pET, pTXB, pUC, and pF3K.
According to claim 1, wherein the promoter is T7 promoter, T5 promoter, T3 promoter, lac promoter, tac and trc promoter, cspA promoter, lacUV5 promoter, Ltet0-1 promoter, phoA promoter, araBAD promoter, trp promoter, tetA promoter, Ptac A production method, characterized in that selected from the group consisting of promoter and SP6 promoter.
The method according to claim 1, wherein the G-tetramerization sequence repeats at least 3 consecutive guanines and 1 to 7 random sequences at least once.
The method according to claim 1, wherein the G-tetramerization sequence comprises SEQ ID NO: 1 (TAGGGTTAGGGT).
A sequence prepared by the method of any one of claims 1 to 7 and complementary to a promoter sequence; a sequence complementary to the target sequence; and a G-quadruplex motif sequence.
A method for producing a hydrogelled nucleic acid comprising the step of amplifying or transcribed from the circular nucleic acid template for producing the hydrogelled nucleic acid of claim 8.
The method according to claim 9, wherein a circular second nucleic acid template comprising a sequence complementary to the promoter sequence, a second target sequence having a length of 30 bp to 120 bp, and a G-quadruplex motif sequence was added. Next, a method for producing a hydrogelled nucleic acid comprising the step of amplification or transcription.
11. The method of claim 10, wherein the second template is added in a molar ratio of 150:1 to 300:1 compared to the circular nucleic acid template (first template) for preparing hydrogelled nucleic acid.
It is prepared by the method of claim 10 and comprises a promoter; target sequence; and a G-tetramerization sequence.
(a') preparing a hydrogelled RNA by transcription (transcription) of the circular nucleic acid template for preparing the hydrogelled nucleic acid of claim 8; and
(b') translating the hydrogelled RNA, a method for producing a peptide or protein comprising the step of synthesizing the peptide or protein.
14. The method of claim 13, wherein step (a') comprises a sequence complementary to the promoter sequence, a second target sequence having a length of 30bp to 120bp, and a G-quadruplex motif sequence. A method for producing a peptide or protein, characterized in that the second nucleic acid template is added and then transcribed to prepare a hydrogelled RNA.
The method according to claim 13, wherein in step (b'), the hydrogelled nucleic acid is pulverized and translated.
The method according to claim 13, wherein in step (b'), the peptide or protein is synthesized through a cell free protein synthesis system.

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