KR20220147264A - Template for Producing Nucleic Acid Hydrogel Based on Various Nucleic Acid Cross-Linking Sequences and Method for Producing Nucleic Acid Hydrogel Using The Same - Google Patents

Abstract

The present invention relates to a nucleic acid template for producing nucleic acid hydrogel and nucleic acid hydrogel using the same. The nucleic acid template for producing nucleic acid hydrogel according to the present invention overcomes the limitation of high costs and a low yield in a currently existing extracellular/intracellular nucleic acid synthesis method, in particular synthesis of RNA and stabilizes the nucleic acid through conversion of the nucleic acid synthesized by using the template into hydrogel, thereby being capable of being useful in a field of a functional RNA-based technology. In particular, the nucleic acid template for producing nucleic acid hydrogel according to the present invention can synthesize nucleic acid hydrogel at low costs with high yields through a circulating nucleic acid synthesis method such as rolling circle amplification (RCA) or rolling circle transcription (RCT). The nucleic acid hydrogel produced with the template of the present invention is very stable due to its unique physical properties and exhibits various characteristics depending on the crosslinking sequence, so that depending on the selection of the crosslinking sequence, the nucleic acid hydrogel can be selected and used very usefully in various industrial fields. In particular, the nucleic acid hydrogel can not only be useful for cell-based or cell-free synthesis of peptides, which requires large-scale synthesis of nucleic acids, but can also be useful in various fields such as biological and medical applications due to high water content, advantageous structural characteristics, and high biocompatibility thereof.

Description

Template for Producing Nucleic Acid Hydrogel Based on Various Nucleic Acid Cross-Linking Sequences and Method for Producing Nucleic Acid Hydrogel Using The Same}

The present invention relates to a nucleic acid template for production of a nucleic acid hydrogel and a nucleic acid hydrogel using the same, and more particularly, a nucleic acid template for production of a nucleic acid hydrogel comprising a sequence complementary to a nucleic acid crosslinking sequence, and the nucleic acid template as a template It relates to a method for producing a nucleic acid hydrogel, characterized in that for synthesizing a nucleic acid.

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 both flows. As research on the flow of genetic information and the function of the composition of the central dogma continues, the synthesis technology of nucleic acids (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. There are disadvantages in that it is difficult to perform an accurate sequencing analysis by finding errors of various factors that occur during nucleic acid sequencing, and 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.

In contrast to DNA, functional single-stranded (e.g. mRNA) and double-stranded RNA molecules have been produced in living cells and in vitro using purified recombinant enzymes and purified nucleotide triphosphates (EP 1631675 and See US Patent Application Publication No. 2014/0271559 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., RNA-based vaccines, siRNA or RNAzyme therapeutics, or miRNA or piwi RNA-based diagnostic platforms, etc.) is being used According to the central dogma that sustains biology, it is produced from DNA to RNA to the final protein. RNA has a very short retention 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, it is difficult to extract or store RNA because the degradation rate of intracellular RNA is faster than that of DNA, 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.

A hydrogel is a supramolecular assembly hosting an aqueous medium, and is characterized by having both the solute transport properties of a liquid and the mechanical properties of a solid. Hydrogels can be particularly useful for biological and medical applications due to their high water content, advantageous structural features and high biocompatibility. The nucleic acid hydrogel obtained by gelling the above nucleic acid has been highlighted for its usefulness as a powerful means of overcoming the instability of nucleic acid, etc., but the existing method such as covalent crosslinking for producing a hydrogel of nucleic acid requires a complicated procedure. There is a disadvantage in that the yield is significantly low compared to the cost. Therefore, despite its usefulness in various industrial fields, the nucleic acid hydrogel has not reached the commercialization stage.

The present inventors, under this background, using a nucleic acid containing a sequence complementary to a G-quadruplex motif sequence as a template Rolling circle transcription (RCT)-based hydrogelling nucleic acid A production method and a protein production system using the hydrogelled nucleic acid prepared by the method were invented and applied for a patent (Korean Patent Application No. 10-2019-0136416, unpublished).

In addition to the G-tetrapolymer-based nucleic acid hydrogel, the present inventors secured various types of sequences capable of forming a nucleic acid hydrogel in addition to the G-tetrapolymer motif sequence, thereby preparing a nucleic acid hydrogel having various mechanical/functional properties As a result of diligent efforts to develop a method, a nucleic acid containing a Prion, BYDV or i-motif forming sequence among the crosslinking sequences known to form a secondary structure that exists in nature or used as an aptamer has been developed through self-assembly. It was confirmed that it is hydrogelled, and it was confirmed that it exhibits higher stability than the present inventor's G-tetrapolymer-based nucleic acid hydrogel, which is the present inventor's invention, and exhibits different physical properties, and completed the present invention.

The above 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 the 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 nucleic acid template for the preparation of a nucleic acid hydrogel.

Another object of the present invention is to provide a composition for producing a nucleic acid hydrogel comprising the nucleic acid template.

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

Another object of the present invention is to provide a nucleic acid hydrogel formed by self-assembly of a nucleic acid crosslinking sequence.

In order to achieve the above object, the present invention provides a nucleic acid template for the production of a hydrogelled nucleic acid comprising a sequence complementary to a nucleic acid crosslinking sequence.

The present invention also provides a composition for producing a nucleic acid hydrogel comprising the nucleic acid template.

The present invention also provides a method for producing a nucleic acid hydrogel comprising the step of synthesizing a nucleic acid using the nucleic acid template as a template.

The present invention also provides a nucleic acid hydrogel prepared by the above preparation method.

The present invention also provides a nucleic acid hydrogel formed by self-assembly of a nucleic acid crosslinking sequence.

The nucleic acid template for the production of hydrogelled nucleic acids of the present invention overcomes the limitations of high cost and low yield in the existing intracellular/extracellular nucleic acid synthesis methods, particularly RNA synthesis, and produces nucleic acids due to hydrogelation of nucleic acids synthesized as templates. By stabilizing it, it can be usefully used in functional RNA-based technical fields. In particular, the template for the production of a nucleic acid hydrogel of the present invention can synthesize a nucleic acid hydrogel in a low cost and high yield through a cyclic nucleic acid synthesis method such as circular cyclic amplification (RCA) or circular cyclic transcription (RCT). The nucleic acid hydrogel prepared from the template of the present invention is very stable due to its unique physical properties, and can be very usefully used in various industrial fields where the hydrogel is used. In particular, it can be usefully used for cell-based or cell-free synthesis of peptides requiring mass synthesis of nucleic acids, and can be usefully used in various fields such as biological and medical applications due to its high water content, advantageous structural characteristics and high biocompatibility. can

1 shows the RNA secondary structure (top) and Gibb's free energy (bottom) between pairs of bridging sequences when various pairs of bridging sequences have spacers of various lengths interposed therebetween.
2 is an electrophoresis result confirming the circular template generation process for circular circular transcription of each template DNA (2a:BYDV-m, 2b:Prion-m, 2c:i-motif). After the ligation process to complete the circular template, the shift of the DNA structure band position on the electrophoresis indicates that ligation has occurred successfully from the point.
Fig. 3 is an image in which various cross-linking sequences generated gels through circular circular transcription with spacers.
4 shows an indentation-force (load) graph obtained through atomic force microscopy (AFM) nanoindentation to confirm the strength of gel formation made from various cross-linking sequences.
5 is a result of confirming the stability of the gel protein expression solution in the case of using a template of the indicated concentration. A darker red color means a harder gel, and a yellow color means the gel is disintegrated.
6 shows the results (upper) and the amount of RNA produced according to the nucleic acid cross-linking sequence (lower) of the gel when i1, i2, i3, and i4 were used as nucleic acid crosslinking sequences by SYBR green II staining.
7 is a result confirming the generation of a gel by treating 1 wt% of 400 Da PEG and 35 kDa PEG together with (pre-treated) and after (post-treated) the circular cyclic transcription process.
8 shows the results of UV spectroscopy of RNA gel post-treated with 35 kDa PEG.
9 is a result of evaluating the stability of the nucleic acid hydrogel in 10% FBS (fetal bovine serum) with or without PEG treatment.

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 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. RNA hydrogel has the advantage that it can overcome the disadvantages of RNA because of its stability due to its unique structure, but its yield is very low despite the complicated production procedure, and the nucleic acid hydrogel prepared by the currently reported manufacturing method The stability, mechanical properties and functional properties of the gel are also insufficient to be utilized in actual industry, and it is difficult to easily control the stability, mechanical properties and functional properties of the nucleic acid hydrogel using the existing production method.

Accordingly, in an embodiment of the present invention, the present inventors have proposed a nucleic acid template comprising a sequence complementary to a crosslinking sequence to determine whether hydrogelation of nucleic acids is possible through various crosslinking sequences known to form secondary structures in living organisms. was designed, and it was confirmed that the RNA hydrogel was formed by self-assembly of the cross-linking sequence included in the nucleic acid produced by transcription of the nucleic acid template as a template. It was confirmed that the inventors' invention exhibits superior stability than the G-tetrapolymer-based nucleic acid hydrogel.

In an embodiment of the present invention, it is confirmed that a nucleic acid hydrogel is generated through self-assembly of crosslinking sequences (Prion, BYDV, i-motif forming sequences and variants thereof) included in the RNA strand generated by transcription of the nucleic acid template as a template. did. In the present invention, the repeated crosslinking sequence included in the nucleic acid strand plays an important role in the assembly of the nucleic acid hydrogel, and the DNA strand formed when the nucleic acid template is transcribed as a template as well as replication or amplification is performed. It can be clearly understood through the description and examples of the present invention that hydrogelation can be achieved by the crosslinking sequence included therein.

Accordingly, in one aspect, the present invention relates to a nucleic acid template for production of a nucleic acid hydrogel comprising a sequence complementary to a nucleic acid crosslinking sequence.

As used herein, the term "nucleic acid crosslinking sequence" refers to an interaction between bases in a nucleic acid crosslinking sequence or an interaction between nucleic acid crosslinking sequences, and is a secondary structure (eg, Stem-loop structure) through self-assembly or a three-dimensional structure between sequences (eg, , refers to a nucleic acid sequence capable of forming a tetrapolymer structure).

In the present invention, the nucleic acid crosslinking sequence is

i) a cap-independent translation elements (CITE) sequence, a fragment or variant thereof;

ii) SEQ ID NO:3 or SEQ ID NO:5; and

iii) it may be characterized in that it comprises a sequence selected from the group consisting of i-motif forming sequences.

In the present invention, the nucleic acid crosslinking sequence may include a cap-independent translation elements (CITE) sequence or a fragment thereof.

In the present invention, the CITE sequence may be characterized in that it forms a secondary structure or a three-dimensional structure between the sequences.

In the present invention, the fragment of the CITE sequence may be, for example, the sequence represented by SEQ ID NO: 2, but is not limited thereto.

The term "CITE (cap-independent translation elements) sequence" of the present invention is an RNA sequence found in the 3'UTR of many RNA plant viruses, and forms a secondary structure in various viruses that do not form a 5'cap to improve mRNA. It has been reported to be stable and may act as a ribosome entry site in some species to mediate translational initiation of proteins (Biochemical Society Transactions. 35 (Pt 6): 1629-1633. doi:10.1042/BST0351629). The sequence of the CITE sequence is very diverse depending on the type of virus, and each is assembled into a different structure, but all of them are characterized by including a sequence capable of stabilizing mRNA by forming a secondary structure such as a stem-loop. The CITE sequence can be classified according to its structural form (Miller et al., 2007). For example, TED (translation enhancer domain) reported in Satellite tobacco necrosis virus (STNV), etc., Barley yellow dwarf virus (BYDV), etc. reported BTE (BYDV-like translation element), Panicum mosaic virus (PMV), etc. reported PTE (PMV-like translation element), Turnip crinkle virus (TCV), etc. reported TSS (T-shaped structure), Y-shaped reported by Tomato bushy stunt virus (TBSV), etc., I-shaped reported by Maize necrotic streak virus (MNeSV), etc., but is not limited thereto (Nicholson, BL; White, KA (Nov 2011)). In one embodiment of the invention, the nucleic acid crosslinking sequence forms Stem-loop I in BTE derived from Barley yellow dwarf virus (BYDV) (Guo et al., 2000), a fragment of the BTE sequence (SEQ ID NO: 2), or It was confirmed that a nucleic acid hydrogel is stably formed even when it includes a sequence (SEQ ID NO: 4) in which some bases of SEQ ID NO: 2 are substituted, and exhibits physical properties different from those containing SEQ ID NO: 2.

In the present invention, the nucleic acid crosslinking sequence may include a sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4.

In the present invention, the nucleic acid crosslinking sequence may include a sequence represented by SEQ ID NO: 3 or SEQ ID NO: 5.

In the present invention, the sequence represented by "SEQ ID NO: 3" (GGAGGAGGAGGA) is self-assembled into an aptamer sequence specific for a soluble normal cellular prion protein (PrP ^c ) to form a G-quadruplex structure. It has been reported to form (Biochimica et Biophysica Acta 1861 (2017) 1429-1447). In an embodiment of the present invention, it was confirmed that a nucleic acid transcribed using a template including a sequence complementary to SEQ ID NO: 3 (SEQ ID NO: 10) as a template stably forms a hydrogel. In addition, it was confirmed that a nucleic acid hydrogel is stably formed even when it includes a sequence (SEQ ID NO: 5) in which some bases of SEQ ID NO: 3 are substituted, and has different physical properties.

In the present invention, the nucleic acid crosslinking sequence may include an i-motif (intercalated motif) sequence.

In the present invention, the i-motif forming sequence may include a sequence represented by SEQ ID NO: 6 to SEQ ID NO: 9.

As used herein, the term “i-motif forming sequence” refers to a cytosine-rich nucleic acid strand, which forms i-motif, a quadruple nucleic acid structure by Hoogsteen base pairs and C:C+ base pairs. The quadruple nucleic acid structure formed by i-motif has a structure similar to that of the G-quartet structure, which is a stable structure formed from a guanine-rich nucleic acid strand. i-motif was first reported to exhibit a quadruplex structure in vitro in 1993 (Nature. 363 (6429): 561-565), and recently has been reported to exist naturally in human cells (Nature Chemistry. 10 (Nature Chemistry. 10 ( 6): 631-637). The quadruplex structure of RNA i-motif can be confirmed in PDB: 1I9K (Journal of Molecular Biology. 309 (1): 139-53). The i-motif forming sequence is a cytosine-rich sequence, and as a representative example, the complementary strand of the G-quadrel motif sequence is also known as the i-motif forming sequence, and an algorithm for determining the i-motif forming sequence has been reported. (Quadparser algorithm, Biochemistry. 56 (36): 4879-4883). The characteristics and formation conditions of the i-motif structure have been studied and reported in detail in the art (Nucleic acids research, 46(16), 8038-8056, etc.).

In the present invention, the i-motif forming sequence may be characterized in that it contains abundant cytosine. For example, the i-motif forming sequence may include one or more C-tracts.

In the present invention, the C-tract refers to a region where two or more cytosines are repeated in a nucleic acid strand. For example, the C-tract may be a sequence in which 2 to 10 cytosines are repeated, but is not limited thereto.

In the present invention, when the i-motif forming sequence includes two or more C-tracts, it is characterized in that it includes bases (eg, A, T, G) except for one or more cytosines between each C-tract. may be, and preferably may be characterized as comprising 1 to 3 thymine (T), but is not limited thereto.

In the present invention, for example, the i-motif forming sequence may be characterized in that the following repeating units are repeated one or more times:

Repeat unit of i-motif forming sequence = (C _n d _m )

In the repeating unit of the i-motif forming sequence, C denotes cytosine, and d denotes a base (A, G, T or U) excluding cytosine.

In the present invention, in the repeating unit of the i-motif forming sequence, n means the number of cytosines, and may be an integer of 2 to 10.

In the present invention, in the repeating unit of the i-motif forming sequence, m means the number of d, and may be an integer of 1 to 3.

In the present invention, the i-motif forming sequence may include a sequence represented by SEQ ID NO: 6 or SEQ ID NO: 7.

In the present invention, the i-motif forming sequence may be characterized in that it is repeated one or more times, for example, it may be characterized as represented by SEQ ID NO: 8 or 9.

In the present invention, examples of the i-motif forming sequence are based on existing reports for determining whether i-motif is formed (Quadparser algorithm, etc.), and the i-motif forming sequence of the present invention is not limited. . In the present invention, the i-motif-forming sequence should be interpreted as meaning including all sequences capable of forming an i-motif.

In the present invention, the nucleic acid crosslinking sequence may be characterized in that it comprises a sequence selected from the group consisting of SEQ ID NOs: 2 to 9.

In the present invention, the sequence complementary to the nucleic acid crosslinking sequence means a sequence complementary to the nucleic acid crosslinking sequence.

For example, in the present invention, the sequence complementary to the nucleic acid crosslinking sequence may include a sequence selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 17.

In the present invention, the sequence complementary to the nucleic acid crosslinking sequence may be included in the nucleic acid template by repeating one or more times, and two or more sequences complementary to different nucleic acid crosslinking sequences may be included.

As used herein, the term “complementary sequence” refers to a sequence capable of hybridizing with a template base sequence. For example, in the present invention, the complementary sequence refers to a sequence capable of complementary hybridization to a specific sequence according to the Watson-Crick base pairing rule (A:T(U), G:C). In the present invention, the complementary sequence is used as a concept including a substantially complementary sequence, and the substantially complementary sequence is sufficient to hybridize both strands under general conditions, for example, nucleic acid hydrogel synthesis conditions, etc. complementary sequence.

As used herein, the term "nucleic acid template" refers to a nucleic acid strand that serves as a template for nucleic acid replication or transcription. In the present invention, the template may be circular or linear, except where specifically mentioned, and the shape may be selected and manufactured by a person skilled in the art if necessary. When the nucleic acid is produced using a rolling circle amplification (RCA) or a rolling circle transcription (RCT) method, it is preferably a circular template. In the case of using the RCA or RCT method using a circular template, the synthesis of nucleic acids is continued until the raw materials (NTP, dNTP, etc.) are exhausted, and it may be characterized in that a high yield is exhibited. In the present invention, the template is a nucleic acid template, for example, may be a DNA or RNA template, preferably a DNA template, but is not limited thereto.

The nucleic acid template for the production of a nucleic acid hydrogel of the present invention is characterized in that when amplification or transcription is performed using the template, the synthesized nucleic acid is hydrogelled to produce a nucleic acid hydrogel.

Therefore, the nucleic acid template for the production of the nucleic acid hydrogel may be characterized in that it further comprises a primer binding site, or a promoter sequence.

In the present invention, the primer binding site refers to an amplification starting point of the nucleic acid template. A primer is bound to the primer binding site, and nucleic acid synthesis and amplification are performed at the 3'-end of the primer.

In the present invention, when the nucleic acid template includes a primer binding site, the nucleic acid hydrogel synthesized from the nucleic acid template may be a DNA hydrogel.

In the present invention, the primer binding site may be designed without limitation to be suitable for amplification using a nucleic acid template as a template.

In the present invention, the promoter sequence is a starting point for producing RNA through transcription, and a suitable promoter is selected 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. and can be used Such promoters include, 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 promoter, Ptac promoter and SP6 promoter and the like, and may preferably be selected from the above examples, but is not limited thereto.

In the present invention, when the nucleic acid template includes a promoter sequence, the nucleic acid hydrogel synthesized from the nucleic acid template may be an RNA hydrogel.

In the present invention, when the nucleic acid template includes a promoter sequence or a primer binding site, the promoter sequence or primer binding site is separated and may be present at the 3'-end and 5'-end of the nucleic acid template (linear). . At this time, as in the embodiment of the present invention, an oligonucleotide comprising a sequence complementary to the promoter sequence or primer binding site is added to the nucleic acid template as the nucleic acid template and annealed, and through ligation, a circular nucleic acid template for RCT or RCA is obtained. Circular nucleic acid templates for making can be prepared.

The term "separation" of a promoter sequence or primer binding site in the present invention means that the promoter sequence or primer binding site is separated into two or more. The separation is possible without restriction of position as long as it is within the promoter sequence or the primer binding site sequence, but for effective annealing with an oligonucleotide comprising a sequence complementary thereto, the promoter sequence present at the 3'-end and 5'-end It is preferable to separate so that the length is similar.

As in an embodiment of the present invention, an oligonucleotide comprising a promoter sequence or a sequence complementary to a primer binding site is annealed to separate promoter sequences or primer binding sites present at the 5'-end and 3'-end of the nucleic acid template. A circular template can be formed by forming a double strand containing nicks or voids, and a complete circular template can be formed by linking nicks or voids by ligation or polymerization. can

In the present invention, the nucleic acid template for the production of the nucleic acid hydrogel may be characterized in that it further comprises a spacer. In the present invention, the spacer may be positioned between each component of the nucleic acid template, for example, a sequence complementary to a nucleic acid crosslinking sequence, and a promoter or primer binding site. In the present invention, the spacer may be designed to be selected to be included in a length sufficient for the preparation of a nucleic acid template, and for designing a nucleic acid template of sufficient size for nucleic acid synthesis. For example, the spacer may be characterized as having a length of 15 to 220 bp, preferably 20-200, 25-150, or 30-120 bp, but is not limited thereto.

In the present invention, the spacer may be characterized in that at least 50% or more, preferably at least 60% or more, more preferably at least 90% or more, most preferably at least 95% or more, is thymine. .

In the present invention, the spacer may be characterized in that all spacers include a poly T sequence composed of thymine, as in an embodiment of the present invention. In the present invention, the spacer may include or consist of 3T, 10T, 30T, 60T, 90T, 120T, or 150T.

In the present invention, the nucleic acid template for the production of the nucleic acid hydrogel further comprises a sequence other than the nucleic acid crosslinking sequence, the promoter sequence, the primer binding site sequence, and / or the spacer according to the use of the nucleic acid hydrogel. can be done with For example, when the nucleic acid hydrogel is used for the production of a polypeptide and/or protein, the nucleic acid hydrogel may further include a coding sequence encoding the polypeptide and/or protein to be produced. The coding sequence of the nucleic acid template is synthesized into a polypeptide or protein through transcription and translation. In the case of producing polypeptides and proteins using the nucleic acid hydrogel prepared based on the nucleic acid template of the present invention, cell-free synthesis is possible, and the synthesis yield is significantly higher at low cost compared to the existing polypeptide synthesis method. It may be characterized by showing (refer to Korean Patent Application No. 10-2019-0136416).

In another aspect, the present invention relates to a composition for producing a nucleic acid hydrogel comprising a nucleic acid template for the production of the nucleic acid hydrogel.

In the present invention, the composition for producing a nucleic acid hydrogel is, in addition to the nucleic acid template for production of a nucleic acid hydrogel of the present invention, another nucleic acid template, a buffer necessary for nucleic acid synthesis, various enzymes, oligonucleotides, NTP, dNTP, rNTP, and/or ddNTP and the like may be further included. In addition, it may include additional components depending on the use of the nucleic acid hydrogel, for example, in the case of a nucleic acid hydrogel for protein or polypeptide synthesis, it may be characterized by additionally including amino acids, ribosomes, and the like.

For a specific example, in the present invention, the composition for producing a nucleic acid hydrogel may further include various enzymes such as nucleic acid polymerase, ligase, and ribosome.

In the present invention, the composition for producing a nucleic acid hydrogel may further include an oligonucleotide. In the present invention, the oligonucleotide may be characterized in that it binds to a nucleic acid template to provide a starting point (eg, 3'-OH) for amplification or transcription.

In another aspect, the present invention provides a method for producing a nucleic acid hydrogel comprising the step of (a) synthesizing a nucleic acid using the nucleic acid template of the present invention as a template.

As used herein, the term "synthesizing a nucleic acid using a nucleic acid template as a template" means that, at the 3'-end of the primer or oligonucleotide bound to the nucleic acid template of the present invention, a nucleotide complementary to the sequence of the nucleic acid template is bound to form a nucleic acid strand. It means stretching along the template.

In the present invention, the step of synthesizing the nucleic acid may be characterized in that amplification or transcription of the nucleic acid template as a template.

As used herein, the term “amplification” refers to synthesizing a nucleic acid strand of the same type as a nucleic acid template serving as a template. For example, when the nucleic acid template of the present invention is a DNA template, it means synthesizing DNA using it as a template, or when the nucleic acid template of the present invention is an RNA template, synthesizing RNA using the nucleic acid template as a template. The amplification of the present invention can be performed without limitation using a variety of methods. For example, through rolling circle amplification (RCA), polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and transcription-mediated amplification (TMA). It can be performed, preferably using RCA, but is not limited thereto. In the present invention, when amplification is performed through rolling circle amplification (RCA), the nucleic acid template may be a circular nucleic acid template. In the present invention, 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.

As used herein, the term “transcription” refers to one of the DNA expression processes. In the present invention, the transcription refers to the synthesis of RNA using the nucleic acid template as a template. The transcription of the present invention can be performed without limitation using various methods, but it is preferable to perform a rolling circle transcription (RCT) method. In the present invention, an appropriate nucleic acid polymerase (Polymerase), a promoter, etc. may be selected or manufactured by a person skilled in the art for the transcription.

In the present invention, the step (a) may be characterized by performing a rolling circle transcription (RCT) or a rolling circle amplification (RCA).

The RCA and RCT refer to performing one-fold or more amplification or transcription using a circular nucleic acid template as a template, and in this case, it is characterized in that the n-fold product is synthesized so as to be continuous as one nucleic acid strand. The RCA and RCT are selectively used according to the type of nucleic acid to be prepared. When a DNA nucleic acid template is used as a template, 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. Therefore, 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 circular transcription or circular circular amplification is used interchangeably in the same meaning as the rolling-circle amplification and the rolling-circle transcription.

In one embodiment of the present invention, it was confirmed that the nucleic acid crosslinking sequence, the length of the included spacer and the concentration of the nucleic acid template used for nucleic acid synthesis exhibit different stability and degradation rate in solution.

In the present invention, the step (a) is preferably characterized in that the nucleic acid is synthesized using a nucleic acid template at a concentration of about 0.25 to about 1.5 μM as a template, but is not limited thereto, and is not limited thereto. Accordingly, it can be synthesized by adjusting the concentration of the nucleic acid template to control the physical, chemical and functional properties of the hydrogel.

In the present invention, the nucleic acid synthesized in step (a) may be characterized in that it forms a hydrogel through self-assembly.

In the present invention, step (a) may be characterized in that it is carried out by adding a salt.

In the present invention, the salt may be selected from the group consisting of, for example, NaCl, MgCl2, KCl and CaCl2, but is not limited thereto.

In the present invention, the salt may be preferably added at a concentration of more than 0 and 400 mM or less, more preferably 50 mM to 150 mM, and most preferably 100 mM.

In the present invention, the conditions for showing stability are different depending on the nucleic acid crosslinking sequence, and those skilled in the art can determine the appropriate length, the nucleic acid crosslinking sequence and the nucleic acid template concentration according to the use and purpose of the nucleic acid hydrogel based on the contents disclosed in the present invention. It can be adjusted to prepare a nucleic acid hydrogel.

In one embodiment of the present invention, when circular circulation transcription is performed by adding polyethylene glycol (PEG) or hyaluronic acid (HA), which are biocompatible, biodegradable high molecular compounds, the stability of the nucleic acid hydrogel in the living environment is significantly improved and , it was confirmed that low cytotoxicity was maintained. Therefore, when nucleic acid is synthesized by adding the polymer compound of the present invention, it is possible to prepare a nucleic acid hydrogel exhibiting properties very suitable for application to a living body.

As confirmed in one embodiment of the present invention, it was confirmed that the addition of the high molecular compound was both treated with the nucleic acid synthesis step or after the nucleic acid synthesis to form a gel.

Therefore, in the present invention, it may be characterized in that the polymer compound is added before step (a), at the same time as step (a) or after step (a) is performed.

In the present invention, the step (a) may be characterized in that it is carried out after adding the polymer compound.

In the present invention, the step (a) may be characterized in that it is performed simultaneously with the addition of the polymer compound.

In the present invention, (b) it may be characterized in that it further comprises the step of reacting by adding a high molecular compound to the nucleic acid synthesized in step (a).

In the present invention, the polymer compound is a biocompatible and/or biodegradable polymer compound, preferably polyethylene glycol (PEG), hyaluronic acid (HA), low melt agarose (LMA), methyl cellulose ( methyl cellulose, MC), nipalm (Poly (N-isopropylacrylamide, NIPAAm), polyvinyl alcohol, sodium polyacrylate, acrylate polymer, collagen, gelatin ( gelatin), and may be characterized as selected from the group consisting of fibrin, more preferably polyethylene glycol (PEG) or hyaluronic acid (HA), but is not limited thereto.

In the present invention, the polymer compound of the present invention preferably has a molecular weight of 1000 kDa or less, more preferably 400 Da to 35 kDa, but is not limited thereto.

In one embodiment of the present invention, the promoter sequence was separated to prepare a linear nucleic acid template comprising a sequence complementary to a nucleic acid crosslinking sequence present at the 3'-end and 5'-end, and the 3'-end of the linear template and annealing an oligonucleotide having a promoter sequence at the 5'-end and a sequence complementary to the promoter sequence to prepare a circular nucleic acid template having a partially double-stranded nick, wherein the nick is ligase ) to prepare a complete circular nucleic acid template by ligation. RCT was performed using the prepared circular nucleic acid template as a template to prepare an RNA hydrogel.

In another aspect, (a) a promoter sequence or primer binding site is separated and a linear template comprising a sequence complementary to a nucleic acid crosslinking sequence present at the 3'-end and 5'-end and the promoter sequence or primer binding annealing an oligonucleotide comprising a sequence complementary to the site; (b) ligating the 3'-end and 5'-end of the annealed nucleic acid template to produce a complete circular nucleic acid template; And (c) relates to a method for producing a nucleic acid hydrogel comprising the step of synthesizing a nucleic acid using the complete circular nucleic acid template as a template.

In the present invention, in step (a), the oligonucleotide is annealed to an isolated promoter sequence or a separate primer binding site present at the 5'-end and 3'-end of the linear nucleic acid template to form a nick or blank It may be characterized in that it forms a circular nucleic acid template comprising a.

In the present invention, step (b) may form a complete circular nucleic acid template by linking nicks or spaces by ligation and/or polymerization.

In the present invention, the synthesis of the nucleic acid in step (c) may be characterized in that it starts at the 3'-end of the annealed oligonucleotide, more preferably the annealed oligonucleotide.

In the present invention, the step (c) may have the same characteristics as described in the "method for preparing a nucleic acid hydrogel comprising the step of synthesizing a nucleic acid using a nucleic acid template as a template".

In the present invention, it may be characterized in that the polymer compound is added before step (c), at the same time as step (c) or after step (c) is performed.

In the present invention, the step (c) may be characterized in that it is performed after adding the polymer compound.

In the present invention, step (c) may be characterized in that it is performed simultaneously with the addition of the polymer compound.

In the present invention, (d) it may be characterized in that it further comprises the step of reacting by adding a polymer compound to the nucleic acid synthesized in step (c). In the present invention, the polymer compound is biocompatible and / or biodegradable high molecular compounds, preferably polyethylene glycol (PEG), hyaluronic acid (HA), low melting point agarose (low melt agarose, LMA), methyl cellulose (methyl cellulose, MC), nipalm (Poly (N) -isopropylacrylamide, NIPAAm), polyvinyl alcohol (Polyvinyl alcohol), sodium polyacrylate, acrylate polymer, collagen, gelatin, and from the group consisting of fibrin (fibrin) It may be characterized as selected, more preferably polyethylene glycol (PEG) or hyaluronic acid (HA), but is not limited thereto.

In the present invention, the polymer compound preferably has a molecular weight of 1000 kDa or less, more preferably 400 Da to 35 kDa.

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

The nucleic acid hydrogel prepared by the method for producing a nucleic acid hydrogel of the present invention may include a nucleic acid crosslinking sequence and may be characterized in that it is gelled by self-assembly of the nucleic acid crosslinking sequence.

In another aspect, the present invention relates to a nucleic acid hydrogel formed by self-assembly of a nucleic acid cross-linking sequence.

The nucleic acid hydrogel of the present invention may be prepared in different sizes and shapes, and may be particularly characterized as exhibiting excellent mechanical properties and physical/chemical stability. The nucleic acid hydrogel of the present invention can be used as a platform for use in a wide range of applications (eg, catalysts, protein expression).

In the present invention, the nucleic acid crosslinking sequence may have the same characteristics as those described in the nucleic acid template for production of a nucleic acid hydrogel of the present invention.

In the present invention, the nucleic acid hydrogel may exhibit different physical/functional properties depending on the nucleic acid crosslinking sequence used.

In the present invention, the nucleic acid hydrogel is formed by interaction between nucleic acid cross-linking sequences. For example, the interaction between the nucleic acid cross-linking sequences is a Watson-Crick base pair bond-based secondary structure formation, Hoogsteen base pair bond-based tetrapolymer structure formation, and antiparallel hairpin-looped duplex formation. and the like, but is not limited thereto.

In the present invention, the nucleic acid hydrogel may be a DNA hydrogel or an RNA hydrogel.

The nucleic acid hydrogel of the present invention may be characterized as having sufficient space inside the gel, and thus has mechanical flexibility. Sufficient space inside the gel can be usefully used for enzymatic reactions (eg, protein synthesis reactions), and when the nucleic acid hydrogel includes a protein-coding region, it can provide sufficient space for ribosomes to access and interact as well. , and thus, it can be usefully used as a cell-free protein production platform and the like. The present inventors have confirmed that, when using a nucleic acid hydrogel containing a nucleic acid cross-linking sequence, the production yield of the polypeptide/protein is significantly improved due to its stability and physical/functional properties.

In the present invention, the nucleic acid hydrogel may include, for example, a functional sequence such as a sequence encoding a polypeptide or a protein.

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: Selection of various nucleic acid cross-linking sequence candidates for preparation of nucleic acid hydrogels and evaluation of secondary structure stability

In order to develop other sequences capable of producing nucleic acid hydrogels other than the G-quartet motif sequence derived from the telomere repeats disclosed in the existing Korean patent applications of the present inventors, it has been reported that the secondary structure of nucleic acids can be formed. Among the RNA sequences, a fragment of the BYDV-like translation element (BTE) sequence, which is a CITE sequence derived from BYDV (Barley yellow dwarf virus) (nucleic acid crosslinking sequence 1, Table 1), a fragment of the prion protein target aptamer sequence (nucleic acid crosslinking sequence 2) , Table 1), sequences modified by substituting some bases of these sequences (BTE, prion aptamer forming sequence) (nucleic acid crosslinking sequences 3 and 4, Table 1), i-motif forming sequences (nucleic acid crosslinking sequences 5 to 8, Tables) 1) was selected as a nucleic acid crosslinking sequence candidate for nucleic acid hydrogelation.

Nucleic acid crosslinking sequence Nucleic acid crosslinking sequence RNA sequence (5'->3') SEQ ID NO: G4 sequence (Control) UAGGGUUAGGGU One Nucleic acid crosslinking sequence 1 (BYDV) GGAUCCUGGGAAACAGGCAGAACUUCGG 2 Nucleic acid crosslinking sequence 2 (Prion) GGAGGAGGAGGA 3 Nucleic acid crosslinking sequence 3 (BYDV-m) GGAUUAAGGGAUUACGGAAUAUUGAUGG 4 Nucleic acid crosslinking sequence 4 (Prion-m) GGUGGUGGUGGU 5 Nucleic acid crosslinking sequence 5 (i1) UACCCUUACCCU 6 Nucleic acid crosslinking sequence 6 (i2) AACCCUAACCCU 7 Nucleic acid crosslinking sequence 7 (i3) UACCCUUACCCUUACCCUUACCCU 8 Nucleic acid crosslinking sequence 8 (i4) AACCCUAACCCUAACCCUAACCCU 9

*Sequence Reference - Nucleic Acid Crosslinking Sequence 1: Wang, Z., et al., Virology 402, 177-186 (2010).

- Nucleic acid crosslinking sequence 2: Platella, C., et al., Biochimica et Biophysica Acta 1861, 1429-1447 (2020).

- Nucleic acid crosslinking sequence 5: Abou Assi, H., et al., Nucleic acids research, 46(16), 8038-8056.

In addition to the nucleic acid crosslinking sequence, additional sequences must be included for the formation of a sufficient length for the formation of a circular template and for the production of a later functional nucleic acid hydrogel (eg, a nucleic acid hydrogel for protein synthesis), so spacers of various lengths are used. It was evaluated whether the secondary structure can be stably formed even when added. The Gibson free energy and secondary structure were predicted using RNAfold webserver [http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi].

As a result, it was confirmed that both the nucleic acid crosslinking sequence 1 (BYDV) and the nucleic acid crosslinking sequence 2 (Prion) form a secondary structure stably even when they include a spacer (up to 300 nt or more) having a length longer than that of G4. Specifically, it was confirmed that nucleic acid crosslinking sequence 1 formed a secondary structure based on Watson-Crick base pair bonding, and nucleic acid crosslinking sequence 2 represents an RNA tetrapolymer form based on Hoogsteen base pair bonding (FIG. 1).

Example 2: Design of nucleic acid template for production of nucleic acid hydrogel and preparation of circular nucleic acid template for RCT

Based on the selected nucleic acid crosslinking sequence, a DNA template for producing a nucleic acid hydrogel containing a sequence complementary thereto was designed. The sequence (DNA sequence) complementary to the nucleic acid crosslinking sequence is shown in Table 2 below.

sequence complementary to the nucleic acid crosslinking sequence Nucleic acid crosslinking sequence name DNA sequence (5'->3') SEQ ID NO: Nucleic acid crosslinking sequence 1 (BYDV) CCGAAGTTCTGCCTGTTTCCCAGGATCC 10 Nucleic acid crosslinking sequence 2 (Prion) TCCTCCTCCTCC 11 Nucleic acid crosslinking sequence 3 (BYDV-m) CCATCAATATTCCGTAATCCCTTAATCC 12 Nucleic acid crosslinking sequence 4 (Prion-m) ACCACCACCACC 13 Nucleic acid crosslinking sequence 5 (i1) AGGGTAAGGGTA 14 Nucleic acid crosslinking sequence 6 (i2) AGGGTTAGGGTT 15 Nucleic acid crosslinking sequence 7 (i3) AGGGTAAGGGTAAGGGTAAGGGTA 16 Nucleic acid crosslinking sequence 8 (i4) AGGGTTAGGGTTAGGGTTAGGGTT 17

In addition to the nucleic acid crosslinking sequence, a nucleic acid template was designed to include spacers of various lengths (30T, 60T, 90T, 120T and 150T), and a promoter sequence for transcription initiation (T7 promoter). A circular nucleic acid template is required to perform circular cyclic transcription, but it is somewhat difficult to produce a circular nucleic acid template including various sequences. Nucleic acid templates were prepared.

In an embodiment of the present invention, the prepared linear nucleic acid template is shown in Table 3 below.

Nucleic Acid Template Nucleic Acid Template DNA sequence (5'->3') SEQ ID NO: 150T-BYDV template ATAGTGAGTCGTATTA CCGAAGTTCTGCCTGTTTCCCAGGATCC (PolyT ATCCCT 18 150T-Prion template ATAGTGAGTCGTATTA TCCTCCTCCTCC (PolyT ATCCCT 19 30T-BYDV-m template ATAGTGAGTCGTATTA CCATCAATATTCCGTAATCCCTTAATCC (PolyT ₃₀ ATCCCT 20 60T-BYDV-m template ATAGTGAGTCGTATTA CCATCAATATTCCGTAATCCCTTAATCC (PolyT ATCCCT 21 90T-BYDV-m template ATAGTGAGTCGTATTA CCATCAATATTCCGTAATCCCTTAATCC (PolyT ₉₀ ATCCCT 22 120T-BYDV-m template ATAGTGAGTCGTATTA CCATCAATATTCCGTAATCCCTTAATCC (PolyT ₁₂₀ ATCCCT 23 150T-BYDV-m template ATAGTGAGTCGTATTA CCATCAATATTCCGTAATCCCTTAATCC (PolyT ATCCCT 24 30T-Prion-m template ATAGTGAGTCGTATTA ACCACCACCACC (PolyT ₃₀ ATCCCT 25 60T-Prion-m template ATAGTGAGTCGTATTA ACCACCACCACC (PolyT ATCCCT 26 90T-Prion-m template ATAGTGAGTCGTATTA ACCACCACCACC (PolyT ₉₀ ATCCCT 27 120T-Prion-m template ATAGTGAGTCGTATTA ACCACCACCACC (PolyT ₁₂₀ ATCCCT 28 150T-Prion-m template ATAGTGAGTCGTATTA ACCACCACCACC (PolyT ATCCCT 30T-i1 ATAGTGAGTCGTATTA AGGGTAAGGGTA (PolyT ₃₀ ATCCCT 30 30T-i2 ATAGTGAGTCGTATTA AGGGTAAGGGTA (PolyT ₃₀ ATCCCT 30T-i3 ATAGTGAGTCGTATTA AGGGTAAGGGTAAGGGTAAGGGTA (PolyT ₃₀ ATCCCT 32 30T-i4 ATAGTGAGTCGTATTA AGGGTT
AGGGTTAGGGTTAGGGTT ( PolyT ₃₀ ATCCCT

* (PolyT _n ) means a Poly T sequence in which thymine (T) is repeated n times

* Underline indicates nucleic acid crosslinking sequence

* Italics indicate isolated promoter sequences

A complete circular nucleic acid template was prepared through hybridization and ligation with an oligonucleotide having a sequence complementary to a promoter sequence using the linear nucleic acid template. Specifically, the prepared linear nucleic acid template was annealed with a primer including a sequence complementary to the T7 promoter sequence to form a circular nucleic acid template including a nick. Annealing was carried out by lowering the temperature to 4°C by cooling at 1°C per minute after 5 minutes at 95°C under the conditions of 45uM DNA template, T7 promoter primer, and 100mM NaCl. The linear nucleic acid template and oligonucleotide (T7 promoter primer) used in the annealing process were each 40 μM in 100 mM NaCl. A circular nucleic acid template (10 μM) containing a nick formed through annealing was ligated at 16°C for 16 hours by adding T4 DNA ligase (Promega M1801). A template was prepared. The volume and buffer of T4 DNA ligase used for ligation were performed according to the protocol of the T4 DNA ligase kit (Promega M1801). Whether the reaction of each step for the preparation of the circular nucleic acid template was successfully performed was confirmed through electrophoresis using a 2% agarose gel at each step (FIG. 2).

Example 3: Synthesis of nucleic acid hydrogel using nucleic acid template for production of nucleic acid hydrogel

The synthesis of the hydrogel using the complete circular nucleic acid template prepared in Example 2 was performed by circular circular transcription using the Hiscribe T7 High Yield RNA Synthesis Kit (New England Biolabs). Specifically, Hiscribe T7 High Yield by adding enzymes, NTPs and buffers (ATP 3μL, UTP 3μL, CTP 3μL, GTP 3μL, 10x buffer 3μL, T7 polymerase 3μL, 1μM template 3μL, DW 9μL) using 1μM complete circular template as a template After transcription according to the protocol provided by the manufacturer of the RNA Synthesis Kit, it was incubated at 37°C. Maximum RNA yield was expected to be reached after 2 h incubation, but persisted for 48 h after transcription initiation.

Example 3-1: Synthesis of a nucleic acid hydrogel based on a CITE sequence, a nucleic acid crosslinking sequence of a prion protein target aptamer, and a mutant sequence thereof

According to the nucleic acid hydrogel synthesis method, CITE sequence (nucleic acid crosslinking sequence 1, Table 1), nucleic acid crosslinking sequence of prion protein target aptamer (nucleic acid crosslinking sequence 2, Table 1) and variant sequences thereof (nucleic acid crosslinking sequence 3 and 4) , Table 1) using a nucleic acid template based on (SEQ ID NOs: 18 to 33), a nucleic acid hydrogel was prepared.

As shown in FIG. 3, a nucleic acid cross-linking sequence 1 (BYDV) and a nucleic acid cross-linking sequence 2 (Prion)-based nucleic acid template (SEQ ID NO: 18 and 19), it was confirmed that a stable nucleic acid hydrogel was produced when nucleic acid was synthesized. In particular, in nucleic acid crosslinking sequence 1 (BYDV) and nucleic acid crosslinking sequence 2 (Prion), the remaining sequences except for the G/C portion that mainly form the secondary RNA structure are arbitrarily substituted so that the G/C content is 10% or less. It was confirmed that nucleic acid templates (SEQ ID NOs: 20 and 29) based on SEQ ID NO: 3 (BYDV-m) and nucleic acid cross-linking sequence 4 (Prion-m) also successfully generated gels.

Next, in order to confirm the strength of gels made from various nucleic acid crosslinking sequences, indentation-force (load) was measured through AFM (atomic force microscopy) nanoindentation. Specifically, the force-indentation curve of the RNA hydrogel is DNP-S probe cantilevers with an aluminum reflux coating (nominal spring constant: 0.12 N/m, tip diameter: 10 nm, driving frequency) : 16-28 kHz) was obtained with a Nanoscope V controller (Bruker Inc.) using ScanAsyst mode. The approach curve was used to measure the Young's modulus of the hydrogel using the Hertzian contact model. 30 μL of the hydrogel was sprayed onto a mica substrate and a force curve was obtained using the peak force tapping mode.

As shown in Figure 4, the elastic modulus of the nucleic acid hydrogel (Poly T ₁₅₀ ) including each nucleic acid crosslinking sequence was different, specifically 53.6 (BYDV), 74.3 (BYDV-m), and 100.1 (Prion- m) was confirmed to have an elastic modulus of Pa. Accordingly, a nucleic acid template including each nucleic acid cross-linking sequence may be appropriately used depending on the field of application and purpose.

Example 3-2: Evaluation of stability in solution of nucleic acid hydrogel based on CITE sequence, nucleic acid crosslinking sequence variant sequence of prion protein target aptamer

Nucleic acid synthesized using nucleic acid templates based on nucleic acid crosslinking sequence variants (nucleic acid crosslinking sequence 3 (BYDV-m) and nucleic acid crosslinking sequence 4 (Prion-m)) including various spacer lengths of SEQ ID NOs: 20 to 29 at various concentrations. The stability of the hydrogel in solution was confirmed. Specifically, the prepared nucleic acid hydrogel was put in a protein expression buffer containing cell extracts, various salts, NTP, amino acids, cysteine, folinic acid, and PEG, and the stability of the nucleic acid hydrogel in solution was confirmed at 1 hour intervals.

As shown in FIG. 5 , it was confirmed that stability was different depending on the length of the spacer included in each nucleic acid crosslinking sequence and the concentration of the nucleic acid template used. Identification of the resulting gel with various stability from the diversified cross-linking sequence indicates that the stability and degradation rate of the nucleic acid hydrogel can be controlled as needed through the selection of the nucleic acid cross-linking sequence, the length of the spacer, and the control of the concentration of the nucleic acid template. .

Example 3-3: Synthesis of nucleic acid hydrogel based on i-motif forming sequence

According to the nucleic acid hydrogel synthesis method, a nucleic acid hydrogel was prepared using a nucleic acid template (SEQ ID NO: 30 to 33) based on an i-motif forming sequence (SEQ ID NO: 6 to 9). Formation of the nucleic acid hydrogel was confirmed by immersing the transcription product in SYBR green II solution and staining to indicate the shape of the gel.

As shown in Figure 6, it was confirmed that the nucleic acid hydrogel was successfully formed in all cases, and it was confirmed that there was some difference in the amount of RNA produced according to the crosslinking sequence.

Example 4: Stabilization of a nucleic acid hydrogel using a polymer compound and characterization of the nucleic acid hydrogel

After confirming that the synthesis of RNA hydrogels exhibiting various characteristics is possible based on various nucleic acid crosslinking sequences, it was confirmed that the gel could be further stabilized through crowding when biocompatible, biodegradable polymers were treated. Formation of a nucleic acid hydrogel was confirmed by treating (pre-treated) or post-treated (post-treated) 1 wt% of 400 Da PEG and 35 kDa PEG. For post-treatment, the RNA hydrogel was treated with the same amount of PEG and incubated for 1 hour.

As shown in Figure 7, it was confirmed that the RNA hydrogel was successfully formed regardless of the treatment period of PEG, and in various cases, the hydrogel was formed regardless of the molecular weight of PEG.

For structural confirmation during PEG treatment, the RNA hydrogel stabilized by post-treatment with PEG of 35 kDa was subjected to UV-visible light spectroscopy at a wavelength of 200-800 nm using Nano Drop 3100c (DE, USA).

As shown in FIG. 8 , unlike the intrinsic peak at 260 nm of the RNA gel not treated with PEG, the peak was weakened after treatment with PEG, confirming that RNA was stably reinforced by PEG.

Example 5: In vivo stability test of nucleic acid hydrogel treated with polymer compound

To determine whether PEG treatment affects in vivo stability, stability in 10% fetal bovine serum (FBS) with or without PEG treatment was evaluated. 100 μL of the RNA hydrogel generated with or without PEG treatment was added to a Petri dish containing 1000 μL of 10% fetal bovine serum (FBS) to create an RNase-rich environment. Decomposition of the nucleic acid hydrogel was visually confirmed, and in order to confirm the total amount of RNA, samples were stored at -20°C at each time interval to minimize the additional effect of serum and annealed for gel electrophoresis analysis.

As shown in Figure 9, in the case of an RNA hydrogel not treated with PEG, it was completely decomposed after 24 hours in the FBS environment and was not visually confirmed, and in the case of a nucleic acid hydrogel treated with PEG, it was maintained more stably in the FBS environment. It was possible to recover the gel even after 24 hours.

The above results mean that the RNA hydrogel of the present invention and the RNA hydrogel produced by processing the polymer compound have very good usability in an in vivo environment.

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> Template for Producing Nucleic Acid Hydrogel Based on Various Nucleic Acid Cross-Linking Sequences and Methods for Producing Nucleic Acid Hydrogel Using The Same <130> P21-B053 <160> 33 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> G-quadruplex sequence <400> 1 uaggguuagg gu 12 <210> 2 <211> 28 <212> RNA <213> Artificial Sequence <220> <223> cross linking sequence 1 (BYDV) <400> 2 ggauccuggg aaacaggcag aacuucgg 28 <210> 3 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> cross linking sequence 2 (Prion) <400> 3 ggaggaggag ga 12 <210> 4 <211> 28 <212> RNA <213> Artificial Sequence <220> <223> cross linking sequence 4 (Prion-m) <400> 4 ggauuaaggg auuacggaau auugaugg 28 <210> 5 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> cross linking sequence 5 (i-motif 1) <400> 5 ggugguggug gu 12 <210> 6 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> cross linking sequence 5 (i-motif 1) <400> 6 uacccuuacc cu 12 <210> 7 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> cross linking sequence 6 (i-motif 2) <400> 7 aacccuaacc cu 12 <210> 8 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> cross linking sequence 7 (i-motif 3) <400> 8 uacccuuacc cuuacccuua cccu 24 <210> 9 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> cross linking sequence 8 (i-motif 4) <400> 9 aacccuaacc cuaacccuaa cccu 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Complementary sequence 1 (BYDV) <400> 10 aacccuaacc cuaacccuaa cccu 24 <210> 11 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Complementary sequence 2 (Prion) <400> 11 tcctcctcct cc 12 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Complementary sequence 3 (BYDV-m) <400> 12 ccatcaatat tccgtaatcc cttaatcc 28 <210> 13 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Complementary sequence 4 (Prion-m) <400> 13 accaccacca cc 12 <210> 14 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Complementary sequence 5 (i1) <400> 14 aggtaaggg ta 12 <210> 15 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Complementary sequence 6(i2) <400> 15 agggttaggg tt 12 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Complementary sequence 7 (i3) <400> 16 agggtaaggg taagggtaag ggta 24 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Complementary sequence 8 (i4) <400> 17 agggttaggg ttagggttag ggtt 24 <210> 18 <211> 200 <212> DNA <213> Artificial Sequence <220> <223> 150T-BYDV template <400> 18 atagtgagtc gtattaccga agttctgcct gtttcccagg atcctttttt tttttttttt 60 ttttttttttt tttttttttt ttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 180 tttttttttt ttttatccct 200 <210> 19 <211> 184 <212> DNA <213> Artificial Sequence <220> <223> 150T-Prion template <400> 19 atagtgagtc gtattatcct cctcctcctt tttttttttt tttttttttt tttttttttt 60 ttttttttttt tttttttttt ttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttat 180 ccct 184 <210> 20 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> 30T-BYDV-m template <400> 20 atagtgagtc gtattaccat caatattccg taatccctta atcctttttt ttttttttt 60 tttttttttt ttttatccct 80 <210> 21 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> 60T-BYDV-m template <400> 21 atagtgagtc gtattaccat caatattccg taatccctta atcctttttt ttttttttt 60 ttttttttttt tttttttttt tttttttttt tttttttttt ttttatccct 110 <210> 22 <211> 140 <212> DNA <213> Artificial Sequence <220> <223> 90T-BYDV-m template <400> 22 atagtgagtc gtattaccat caatattccg taatccctta atcctttttt ttttttttt 60 ttttttttttt tttttttttt ttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt ttttatccct 140 <210> 23 <211> 170 <212> DNA <213> Artificial Sequence <220> <223> 120T-BYDV-m template <400> 23 atagtgagtc gtattaccat caatattccg taatccctta atcctttttt ttttttttt 60 ttttttttttt tttttttttt ttttttttt tttttttttt tttttttttt tttttttttt 120 ttttttttttt tttttttttt tttttttttt tttttttttt ttttatccct 170 <210> 24 <211> 200 <212> DNA <213> Artificial Sequence <220> <223> 150T-BYDV-m template <400> 24 atagtgagtc gtattaccat caatattccg taatccctta atcctttttt ttttttttt 60 ttttttttttt tttttttttt ttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 180 tttttttttt ttttatccct 200 <210> 25 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> 30T-Prion-m template <400> 25 atagtgagtc gtattaacca ccaccacctt tttttttttt tttttttttt ttttttttat 60 ccct 64 <210> 26 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> 60T-Prion-m template <400> 26 atagtgagtc gtattaacca ccaccacctt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt ttttttttat ccct 94 <210> 27 <211> 124 <212> DNA <213> Artificial Sequence <220> <223> 90T-Prion-m template <400> 27 atagtgagtc gtattaacca ccaccacctt tttttttttt tttttttttt tttttttttt 60 tttttttttt tttttttttt ttttttttt tttttttttt tttttttttt ttttttttat 120 ccct 124 <210> 28 <211> 154 <212> DNA <213> Artificial Sequence <220> <223> 120T-Prion-m template <400> 28 atagtgagtc gtattaacca ccaccacctt tttttttttt tttttttttt tttttttttt 60 ttttttttttt tttttttttt ttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt ttttttttat ccct 154 <210> 29 <211> 184 <212> DNA <213> Artificial Sequence <220> <223> 150T-Prion-m template <400> 29 atagtgagtc gtattaacca ccaccacctt tttttttttt tttttttttt tttttttttt 60 ttttttttttt tttttttttt ttttttttt tttttttttt tttttttttt tttttttttt 120 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttat 180 ccct 184 <210> 30 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> 30T-i1 template <400> 30 atagtgagtc gtattaaggg taagggtatt tttttttttt tttttttttt ttttttttat 60 ccct 64 <210> 31 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> 30T-i2 template <400> 31 atagtgagtc gtattaaggg taagggtatt tttttttttt tttttttttt ttttttttat 60 ccct 64 <210> 32 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> 30T-i3 template <400> 32 atagtgagtc gtattaaggg taagggtaag ggtaagggta tttttttttt tttttttttt 60 tttttttttt atccct 76 <210> 33 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> 30T-i4 template <400> 33 atagtgagtc gtattaggg ttagggttag ggttagggtt tttttttttt tttttttttt 60 tttttttttt atccct 76

Claims (20)

A nucleic acid template for the production of a nucleic acid hydrogel comprising a sequence complementary to a nucleic acid crosslinking sequence,
The nucleic acid crosslinking sequence is
i) a cap-independent translation elements (CITE) sequence, a fragment or variant thereof;
ii) SEQ ID NO:3 or SEQ ID NO:5; and
iii) a nucleic acid template selected from the group consisting of i-motif forming sequences.
The nucleic acid template according to claim 1, which is a circular nucleic acid template.
According to claim 2, wherein the CITE (cap-independent translation elements) sequence is Satellite tobacco necrosis virus (STNV) derived from TED (translation enhancer domain), Barley yellow dwarf virus (BYDV) derived from BTE (BYDV-like translation element) , PTE (PMV-like translation element) from Panicum mosaic virus (PMV), TSS (T-shaped structure) from Turnip crinkle virus (TCV), Y-shaped from Tomato bushy stunt virus (TBSV), and Maize necrotic A nucleic acid template selected from I-shaped from streak virus (MNeSV).
The method of claim 2, wherein the i-motif forming sequence comprises a repeating unit one or more times,
The repeating unit is (Cndm),
Wherein n is an integer of 2 to 10,
Wherein m is a nucleic acid template, characterized in that it is an integer of 1 to 3.
The nucleic acid template according to claim 1, wherein the sequence complementary to the nucleic acid crosslinking sequence comprises a sequence selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 17.
The nucleic acid template according to claim 1, further comprising a primer binding site or a promoter sequence.
According to claim 6, wherein the promoter sequence 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, A nucleic acid template selected from the group consisting of a Ptac promoter and an SP6 promoter.
7. The method of claim 6, wherein the nucleic acid template is a linear nucleic acid template,
The nucleic acid template, characterized in that the primer binding site or promoter sequence is separated and located at the 3'-end and 5'-end of the linear nucleic acid template.
The nucleic acid template of claim 8 , wherein the nucleic acid template is annealed with an oligonucleotide comprising a sequence complementary to a primer binding site or a promoter sequence to form a circular template comprising a nick or a blank.
The nucleic acid template of claim 1, further comprising a spacer.
The nucleic acid template according to claim 10, wherein the spacer is 15 to 200 bp in length.
A composition for preparing a nucleic acid hydrogel comprising the nucleic acid template of any one of claims 1 to 11.
13. The composition of claim 12, further comprising an oligonucleotide comprising a sequence complementary to a primer or a promoter sequence.
A method for producing a nucleic acid hydrogel comprising the step of synthesizing a nucleic acid using the nucleic acid template of claim 1 as a template.
15. The method of claim 14, wherein the synthesizing of the nucleic acid comprises performing a rolling circle amplification (RCA) or a rolling circle transcription (RCT).
The method of claim 15, further comprising the step of reacting by adding a polymer compound.
The method according to claim 16, wherein the step of adding and reacting the polymer compound is performed before the step of synthesizing the nucleic acid, simultaneously with the step of synthesizing the nucleic acid or after the step of synthesizing the nucleic acid. .
The method of claim 16, wherein the high molecular compound is polyethylene glycol (PEG), hyaluronic acid (HA), low melting point agarose (low melt agarose, LMA), methyl cellulose (methyl cellulose, MC), nipalm (Poly (N) -isopropylacrylamide, NIPAAm), polyvinyl alcohol (Polyvinyl alcohol), sodium polyacrylate, acrylate polymer, collagen, gelatin, and from the group consisting of fibrin (fibrin) Method for producing a nucleic acid hydrogel, characterized in that selected.
(a) a linear template comprising a sequence complementary to a nucleic acid crosslinking sequence present at the 3'-end and 5'-end by separating the promoter sequence or primer binding site, and a sequence complementary to the promoter sequence or the primer binding site annealing the oligonucleotide;
(b) ligating the 3'-end and 5'-end of the annealed nucleic acid template to produce a complete circular nucleic acid template; and
(c) a method for producing a nucleic acid hydrogel comprising the step of synthesizing a nucleic acid using the complete circular nucleic acid template as a template.
A nucleic acid hydrogel formed by self-assembly of nucleic acid crosslinking sequences,
The nucleic acid crosslinking sequence is
i) a cap-independent translation elements (CITE) sequence, a fragment or variant thereof;
ii) SEQ ID NO:3 or SEQ ID NO:5; and
iii) a nucleic acid hydrogel selected from the group consisting of i-motif forming sequences.

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