KR20240172319A - Dumbbell-type template with transformed structure and rolling circle amplification using the same - Google Patents

Abstract

The present invention relates to a dumbbell-shaped template with a transformed structure and an isothermal amplification reaction using the same. The dumbbell-shaped nucleic acid template of the present invention has one or more arm structures and a cleavage enzyme recognition site, so that multiple target genes can be detected specifically and efficiently.

Description

{DUMBBELL-TYPE TEMPLATE WITH TRANSFORMED STRUCTURE AND ROLLING CIRCLE AMPLIFICATION USING THE SAME}

The present invention relates to a dumbbell-shaped template with a transformed structure and an isothermal amplification reaction using the same, and more specifically, the present invention relates to a dumbbell-shaped nucleic acid template having one or more arm structures and having a specific position of a cleavage enzyme, thereby improving the efficiency of an isothermal amplification reaction. When an isothermal amplification reaction is performed using the nucleic acid template of the present invention, multiple target genes can be efficiently detected.

Due to population growth and aging, the need to diagnose diseases more precisely and establish treatment strategies accordingly is gradually increasing. Nucleic acid amplification technology is being used as a diagnostic technology such as biomarkers for this purpose, and among them, the polymerase chain reaction (PCR), which is the most widely used, can amplify a specific target genetic material in a large amount in a short period of time, and is used to detect and diagnose infectious diseases, genetic diseases, etc. However, despite these advantages, general PCR requires expensive equipment such as a thermocycler because it amplifies target nucleic acids by controlling the reaction temperature (95℃-55℃-70℃) dozens of times or more.

The isothermal amplification method, which has been suggested as an alternative to the above problems, is amplified at isothermal temperature without the need to change the temperature, so expensive equipment such as a temperature control device is not required. Due to this advantage, it can be utilized in field diagnosis that is difficult to implement with general PCR. Among the isothermal amplification methods, the rolling circle amplification (RCA) technology has recently been in the spotlight. The rolling circle amplification technology can be performed under isothermal conditions at room temperature (37℃), so it is easy to utilize in field diagnosis, etc. In addition, since the target nucleic acid is generated as a long single-stranded DNA, a signal amplified more than 1000 times can be obtained, so the target nucleic acid can be detected with high sensitivity even when there is confusion due to the background signal. In addition, unlike PCR reactions, it can be amplified not only in a solution state but also in a state fixed to the surface of glass, microwell plates, beads, cells, etc., so it can be widely applied to not only the diagnosis of infectious diseases by diagnosing DNA and RNA of viruses and bacteria using existing PCR, but also the signal amplification of FISH and Immunoassay-based experiments, and the detection of intracellular proteins and genomes, and the fields of diagnosis and biosensing, genomics, and proteomics.

Isothermal rolling circle amplification (RCA) technology is one of the isothermal amplification techniques that simply and efficiently generates long-stranded ssDNA or ssRNA using DNA and RNA polymerases. RCA technology has been widely used not only in the diagnostic field but also in cloning and sequencing, and recently, it has been used as a tool for various biological and nanomedical applications such as drug delivery, electronic circuit manufacturing, biosensing, and bioimaging through DNA self-assembly and nanostructure formation.

In particular, in RCA technology, nucleotides are continuously added to a primer (or target gene) hybridized to a circular template to generate a long DNA or RNA strand with a length of tens to thousands and a sequence complementary to the template DNA. Therefore, various application studies can be performed, such as producing DNA (e.g. DNA aptamer) with a desired base sequence through custom design (base sequence or structure) of template DNA, increasing amplification efficiency by including the action sequence of a restriction enzyme, and inducing a selective amplification reaction by causing a structural change by a specific external stimulus.

In the case of the dumbbell template that causes the existing structural change, it has one stem and two loop sequences and provides a toehold site in one loop to form a circular template through the toehold-mediated strand displacement mechanism by the target gene and initiate RCA. A method of controlling the length of the toehold is used to control the RCA amplification efficiency.

Therefore, a new dumbbell template design is required to dramatically improve the RCA amplification efficiency of the existing dumbbell template and to apply additional modified RCA (e.g., NickRCA) using restriction enzymes. In particular, in the case of NickRCA, since it has an inhibitory effect on the NickRCA reaction due to the competitive reaction between DNA polymerase and nicking enzyme, a method to increase the NickRCA efficiency through a new dumbbell template design is required.

The present invention aims to provide a nucleic acid template having a multi-arm structure that enables multiple detection of target genes (multi-primed RCA possible).

The present invention also seeks to provide a nucleic acid template that can obtain an improved fluorescence signal compared to a conventional dumbbell template during an isothermal amplification reaction and can increase amplification efficiency by reducing the competitive reaction between a gene polymerase and a cleavage enzyme during an isothermal amplification reaction.

In addition, the present invention aims to provide a nucleic acid template that can directly induce hybridization and RCA between target genes without a ligation process, thereby performing an isothermal amplification reaction in one step.

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

The present invention is designed to solve the above problem, and provides a dumbbell-shaped rolling circle amplification reaction nucleic acid template, which comprises at least one gene sequence of an arm structure, wherein the gene sequence of the arm structure (hereinafter referred to as “arm”) is composed of a stem sequence and a loop sequence, and a toehold site and a cleavage recognition site are located on the gene sequence of the arm structure.

The recognition site of the cleavage enzyme of the present invention may be present on a loop sequence, and may be located on a sequence 1 to 15 nt apart from the toehold site or on a separate loop.

The template of the present invention has a length of 100 to 150 nt, and the stem sequence can have a length of 10 to 30 bp.

The loop sequence of the present invention may have a length of 15 to 45 bp, and it is preferable that the nucleic acid template of the present invention is composed of a gene sequence (hereinafter, “arm”) having a 2 or 3 arm structure.

The above-described toehold portion of the present invention may have a length of 10 to 30 bp, and the above-described cleavage enzyme recognition portion may have a length of 10 to 30 bp, but is not limited thereto.

The nucleic acid template of the present invention may be comprised of any one of the gene sequences represented by SEQ ID NOs: 2 to 9, but is not limited thereto.

The present invention also provides a composition for a rolling circle amplification reaction comprising any one of the above nucleic acid templates.

The present invention also provides a method for detecting a target gene using the nucleic acid template, comprising the steps of: a) introducing a sample into a composition comprising the nucleic acid template of claim 1, a primer, and a cleavage enzyme; and b) performing a rolling circle amplification reaction.

Among the target gene detection methods of the present invention, step b) may include, when a target gene is present in a sample, i) a step in which the target gene and a primer bind to a nucleic acid template; and ii) a step in which a gene polymerase binds, the dumbbell-shaped nucleic acid template is released, and a rolling circle amplification reaction proceeds.

After step b) of the present invention, a step c) of adding a fluorescent substance capable of binding to the product according to step b) and determining that the target substance is present when fluorescence appears may be further included.

The present invention uses a template having a three-arm structure (triple arm dumbbell template (DB3)) compared to a conventional dumbbell template (single arm dumbbell template (DB 1)), and thus can have sites to which one to three or more target genes can bind, enabling multiple detection of target genes (multi-primed RCA possible).

In addition, since the nucleic acid template of the present invention has multiple arm structures compared to the existing dumbbell template (DB1, (about 56 nt)), it is longer (about 147 nt) and there are about 3 stem regions forming double strands per template, so that the dye can be incorporated into the DNA product obtained through RCA with higher efficiency when SYBR Green fluorescence is stained. Accordingly, a higher fluorescence signal can be obtained, and thereby improved RCA amplification efficiency can be expected.

The nucleic acid template of the present invention is in a form capable of self-ligation without a primer, and the nucleic acid template of the present invention can be included in a master mix together with other RCA reagents (enzyme, dNTP, reaction buffer, etc.).

In addition, the nucleic acid template of the present invention has an advantage in that, compared to other RCA processes that undergo a two-step reaction in which a template and a target gene undergo hybridization and ligation before an RCA reaction, the nucleic acid template of the present invention can directly induce hybridization and RCA between target genes without a ligation process. That is, the present invention can cause a reaction in one step, thereby reducing unnecessary processes.

FIG. 1 is a design diagram of a template having a dumbbell-shaped structure according to one embodiment of the present invention, and the template of FIG. 1 is composed of one arm, one stem, and two loop structures.
FIG. 2 is a schematic diagram of a nucleic acid template according to one embodiment of the present invention, which is composed of two arm structures, two stem structures, and two loop structures.
FIGS. 3 to 6 are schematic diagrams of nucleic acid templates according to one embodiment of the present invention, each of which consists of three arm, three stem, and three loop structures. FIG. 3 has a toehold region of 10 nt in length, FIG. 4 has 11 nt, FIG. 5 has 12 nt, and FIG. 6 has 13 nt, each of which can bind to three different target genes, and the lengths of the stem gene sequences are designed to be different from each other.
FIG. 7 shows a structure of a nucleic acid template designed according to one embodiment of the present invention, which is composed of one arm, one stem, and two loop structures, and it can be seen that a cleavage enzyme recognition site is located on a loop sequence, and a target binding site is located on a connected sequence including a part of the loop sequence and a part of the stem sequence.
FIG. 8 is a schematic diagram of a nucleic acid template according to one embodiment of the present invention, which is composed of three arm, three stem, and three loop structures. In FIG. 8, the cleavage enzyme can be positioned on any one of the loop sequences.
FIG. 9 is a result of electrophoresis (15% dPAGE) according to one embodiment of the present invention. In this embodiment, the ends of a linear template manufactured to form a nucleic acid template having a cancer structure of the present invention were ligated (primer-free self ligation) using T4 DNA, and then treated with exonuclease I/III to remove the unligated free linear template. Through the Denatured PAGE result of FIG. 9, it can be confirmed that the ligation was successful and a dumbbell-shaped nucleic acid template that is not degraded by exonuclease was manufactured.
Figure 10 is a result of electrophoresis (10% nPAGE) according to one embodiment of the present invention. In this embodiment, for each template, hybridization with the target gene (miR-21) was analyzed through native PAGE. Referring to Figure 10, it can be confirmed that hybridization with the target gene occurs in all templates, and in particular, it can be confirmed that hybridization with the target gene occurs better as the toehold region in the DB3 template becomes longer.
FIG. 11 shows the results of performing a rolling circle amplification (RCA) reaction at different temperatures using a nucleic acid template (DB3 T13 N1) manufactured according to one embodiment of the present invention. Referring to FIG. 11, it can be confirmed that the difference in fluorescence signals for the presence or absence of a target gene is most distinct when the RCA reaction temperature is 50°C. Therefore, the optimal reaction temperature in an RCA experiment using the DB3 nucleic acid template of the present invention is about 40 to 60°C, preferably 50°C.
FIG. 12 shows the results of performing a rolling circle amplification (RCA) reaction using nucleic acid templates (DB3 T13 N1, DB1 T10 N1, DB2 T10 N1) having different toehold portion lengths, manufactured according to one embodiment of the present invention. Referring to FIG. 12, it can be seen that DB3 T13 N1 has higher amplification efficiency than DB1 T10 N1 and DB2 T10 N1. That is, it can be seen that as the toehold length in the DB3 nucleic acid template increases, the RCA amplification efficiency increases.
FIG. 13 shows the results of performing a rolling circle amplification (RCA) reaction using two nucleic acid templates including/excluding a cleavage enzyme recognition site, manufactured according to one embodiment of the present invention. In the case of DB1 T10 N1, it can be confirmed that the fluorescence is lower in the nucleic acid template (Nt template) in which the cleavage enzyme recognition site is present, and in the case of DB3 T13 N1, the fluorescence is higher in the Nt template. In other words, it can be seen that the nucleic acid template (Nt template) in which the cleavage enzyme recognition site is present is more suitable for taking advantage of the triple-arm dumbbell nucleic acid template.
FIG. 14 shows the results of performing Nick RCA using a nucleic acid template manufactured according to one embodiment of the present invention. When a Nick RCA reaction is induced using a nicking enzyme, it can be confirmed that fluorescence increases exponentially compared to the existing RCA. Referring to the contents of FIG. 14, in the case of the DB1 T10 N1 (Nt) template, it can be confirmed that exponential amplification occurs in a short period of time even in the absence of a target nucleic acid, resulting in a nonspecific reaction. On the other hand, in the case of the DB3 T10 N2 (Nt) template, it is difficult to confirm a nonspecific reaction like the DB1 T10 N1 (Nt) template. That is, it can be seen that when DB3 is used according to the present invention, not only does it increase both the RCA or Nick RCA efficiency compared to DB1, but it is also effective in reducing the nonspecific reaction observed in Nick RCA.
FIG. 15 shows the results of 10% nPAGE analysis to analyze each product after performing a rolling circle amplification (RCA) reaction at different temperatures using nucleic acid templates (DB1 T10 N1, DB3 T13 N1) manufactured according to one embodiment of the present invention. It can be confirmed that in the case of DB3, there is a clear difference in the amount of DNA product depending on the presence or absence of a target gene in the NickRCA reaction compared to DB1.

Hereinafter, the present invention will be described in detail. The advantages and features of the present invention and the embodiments that achieve them will be apparent by referring to the embodiments described below. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms, and these embodiments are provided only to make the disclosure of the present invention complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims. Like reference numerals refer to like elements throughout the specification.

Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used in a meaning that can be commonly understood by a person of ordinary skill in the art to which the present invention belongs. In addition, terms defined in commonly used dictionaries shall not be ideally or excessively interpreted unless explicitly specifically defined. The terminology used in this specification is for the purpose of describing embodiments and is not intended to limit the present invention. In this specification, the singular also includes the plural unless specifically stated in the phrase.

The present inventors have conducted research efforts to develop a nucleic acid template (template template) capable of efficiently detecting various target genes through a rolling circle amplification method, and as a result, have confirmed that a plurality of target nucleic acids can be detected with high efficiency when rolling circle amplification is performed using a template manufactured to have a plurality of arm structures (or shapes), thereby completing the present invention.

Accordingly, the present invention provides a dumbbell-shaped nucleic acid template for rolling circle amplification reaction, which comprises at least one arm consisting of a stem sequence and a loop sequence; and a toehold site and a cleavage enzyme recognition site on the arm sequence; and a rolling circle amplification reaction using the same.

The term "target gene" used in the present invention means all kinds of genes to be detected, and includes gene base sequences derived from different species, subspecies, or variants, or gene mutations within the same species. It can be characterized by, but is not limited to, all kinds of DNA including genomic DNA, mitochondrial DNA, and viral DNA, or all kinds of RNA including mRNA, ribosomal RNA, non coding RNA, tRNA, viral RNA, etc., and can be characterized by, but is not limited to, a mutant base sequence including a variation in base sequence, and the mutation can be characterized by being selected from the group consisting of single nucleotide polymorphism (SNP), insertion, deletion, point mutation, fusion mutation, translocation, inversion, and LOH (loss of heterozygosity), but is not limited thereto.

The term "target gene" as used herein refers to a nucleotide polymer, and unless otherwise limited, includes known analogs of natural nucleotides that can act in a similar manner (e.g., hybridize) to naturally occurring nucleotides. Target genes of the present invention include, for example, genomic DNA; complementary DNA (cDNA) (which is the DNA representation of mRNA, usually obtained by reverse transcription or amplification of messenger RNA (mRNA)); synthetically or amplified DNA molecules; and any form of DNA or RNA, including mRNA, including single-stranded molecules as well as double- or triple-stranded nucleic acids. In a double- or triple-stranded nucleic acid, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).

The "target gene" of the present invention also includes any chemical modification thereof, such as by methylation and/or capping. Nucleic acid modifications may include the addition of chemical groups, including additional charge, polarizability, hydrogen bonding, electrostatic interactions, and functionality, to individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications, such as modifications at the 2' position, modifications at the 5' position pyrimidine, modifications at the 8' position purine, modifications at the cytosine exocyclic amine, substitution of 5-bromo-uracil, backbone modifications, special base pairing combinations such as the isobaric bases isocytidine and isoguanidine, and the like.

The "target gene" of the present invention may be derived from a completely chemical synthetic process, such as solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acids, or from a process involving handling of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of these processes.

The term "nucleic acid template" used in the present invention refers to a template (probe) designed complementary to a target sequence for rolling circle amplification, also called circularizing oligonucleotide probes, and is composed of a circular loop sequence; and stem sequences that extend from each loop sequence to form a double strand and are each connected to each other. One loop sequence and a stem sequence extending from the loop sequence are called an arm structure, and in the gene sequence of one arm structure (form), one loop sequence and one stem sequence are generally present, but structurally, only one stem sequence may be present to connect two loop sequences as an exception (see FIG. 1). In the present invention, the gene sequence at the position where the loop sequence and the stem sequence are connected, that is, the position including a part of the loop sequence and a part of the stem sequence, may be composed of a sequence specific to the target gene (or primer). Among the sequences specific to the above target gene, the portion corresponding to the loop sequence is called a toehold region. The nucleic acid template of the present invention can be circularized with the help of a ligase during the rolling circle amplification process.

The nucleic acid template of the present invention may have one or more arm structures, preferably two or three arm structures (see FIGS. 2 to 6 ). Most preferably, it has three arm structures.

The nucleic acid template of the present invention also includes a nicking enzyme recognition site within the loop sequence. NickRCA includes a nicking site for a specific nicking enzyme in the template, and a portion of the complementary base sequence generated through RCA is nicked by the nicking enzyme to form a nicked site. At this time, the 3'OH at the end of the nicked site formed is newly recognized by DNA polymerase to initiate a polymerization reaction, and multiple polymerases can act on one template to induce a chain amplification reaction.

Therefore, in the case of general NickRCA using a nicking enzyme, DNA polymerase and the nicking enzyme compete with each other for one template and perform DNA polymerization and nicking at the same time, and this competitive reaction may inhibit the amplification efficiency of NickRCA. However, the nucleic acid template of the present invention has two or three or more arm structures (if the size of the circular template DNA is large (DB3 > DB1), the physical distance between the two enzymes can be increased) and has a size of 100 to 150 nt (preferably 120 to 150 nt, more preferably 147 nt), so that the competitive reaction between the two enzymes, DNA polymerase and the nicking enzyme, can be inhibited, and thus the NickRCA efficiency can be increased.

The length of the base sequence of the nucleic acid template provided in the present invention is as follows.

The toehold region of the present invention has a length of 10 to 30 bp, and preferably has a length of 10 to 13 bp or 20 to 30 bp. The position on the template to which the primer or target gene of the present invention binds includes a toehold and stem region. Depending on the length of the target gene, the entire base sequence may be fully hybridized or partially hybridized.

The stem sequence of the present invention has a length of 10 to 30 bp, preferably 12 to 15 bp or 20 to 30 bp.

The loop sequence of the present invention has a length of 15 to 45 bp, preferably 16 to 26 bp or 10 to 45 bp.

The nicking site of the present invention has a length of 10 to 30 bp. The nicking site of the present invention is included in a loop base sequence and may be located at a distance of 1 to 10 nt from a toehold base sequence, or may be included in a base sequence of a loop different from a loop in which a toehold base sequence exists.

In the present invention, the nucleic acid template may include a marker portion, and a fluorescent probe including a fluorescent substance and DNA may be bound to the marker portion, and the fluorescent substance included in the fluorescent probe may be a fluorescent donor or a fluorescent acceptor.

The fluorescent materials include, but are not limited to, Alexa Fluor 350, Cascade Blue, Alexa Fluor 405, Alexa Fluor 430, nitrobenzoxadiazole (NBD), fluorescein, Alexa Fluor 488, Oregon Green 488, BODIPY 493/503, Rhodamine Green, BODIPY FL, Oregon Green 514, Alexa Fluor 514, Eosin, Rhodamine 6G, BODIPY R6G ...488, Oregon Green 488, BODIPY 493/503, Rhodamine Green, BODIPY FL, Oregon Green 514, Alexa Fluor 514, Eosin, Rhodamine 6G, BODIPY R6G, Alexa Fluor 488, Oregon Green 488, BODIPY 493/503, Rhodamine Green, BODIPY FL, Oregon Green 514, Alexa Fluor 514, Eosin, Rhodamine 6G, BODIPY R6G, Alexa Fluor 488, Oregon Green 488, BODIPY 493/503, Rhodamine Green, BODIPY FL, Oregon Green 514, BODIPY 493/503, Rhodamine Green, BODIPY FL, Oregon Green 514, BODIPY 493/503, Rhodamine Green, BODIPY FL, Oregon Green Alexa Fluor 532, BODIPY 530/550, BODIPY TMR, Alexa Fluor 555, tetramethyl rhodamine (TMR), Alexa Fluor 546, BODIPY 558/568, QSY 7, QSY 9, BODIPY 564/570, Lissamine rhodamine B, Rhodamine Red, BODIPY 576/589, Alexa Fluor 568, X-rhodamine, BODIPY 581/591, BODIPY TR, Alexa Fluor The compound may be at least one selected from the group consisting of Alexa Fluor 594, Texas Red, Alexa Fluor 610-X, BODIPY 630/650, Alexa Fluor 633, Alexa Fluor 635, BODIPY 650/665, Alexa Fluor 647, QSY 21, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, and ATTO, but is not limited thereto.

The terms expressing the nucleic acid templates described in the present invention can be understood as shown in Table 1 below.

DB# T# N# detail DB# Dumbbell template # = 1, 2, 3, etc., depending on the number of arms T# Toehold site # = According to the number of bases in the toehold site N# Nicking site # = According to the number of nicking enzyme recognition sites

In addition, in the present invention, the nucleic acid template according to the number of arms can be designed as shown in Table 2 below. It is not limited to the contents presented in Table 2.

Number of arms Stem and loop sequences One (Single arm template) One stem sequence and two loop sequences Two (Dual arm template) Two stem sequences and two loop sequences Three (Triple arm template) Three stem sequences and three loop sequences

Hereinafter, in order to help understand the present invention, examples will be given and described in detail. However, the following examples are only intended to illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.

Production of nucleic acid templates

In this example, after producing a linear template, both ends were ligated (primer-free self ligation) using T4 DNA ligase, and exonuclease I/III was treated to remove any remaining linear template that was not ligated after ligation, thereby producing a dumbbell-shaped nucleic acid template. In detail, 5' phosphorylated linear template DNA (4 μM) was annealed by heating at 70°C for 10 minutes in 1X T4 DNA ligation buffer (50 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM ATP, 10 mM DTT, pH 7.5) and slowly cooling to 25°C. 2 μL of T4 DNA ligase (16 U/μL) was added, and the ligation reaction to link the nick site formed between the 5' phosphate group and the 3' hydroxyl group was performed at 25°C for 2 h, followed by heating at 65°C for 10 min to inactivate T4 DNA ligase. After that, to remove the unligated linear template DNA, the ligated template DNA was treated with Exonuclease I/III in 1X exonuclease reaction buffer (10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 1 mM DTT, pH 7) at 37°C for 1 h, and then inactivated by heating at 80°C for 15 min. The produced dumbbell-shaped template was stored at -20°C and used in the subsequent RCA reaction.

The gene sequences used in the present invention are as shown in Table 3 below.

designation Sequence (5' →3') Sequence number miR-21 (target) UAGCUUAUCAGACUGAUGUUGA 1 DB1 T10 N1 /5Phos/ AAGCTA TTGTAATACCTCAGCC TAGCTTATCAGA GCCCCC TCAACATCAG TCTGAT 2 DB2 T10 N1 /5Phos/ TAGCTTATCAGA GCCTTCAGCCTCAACATCAG TCTGATAAGCTATAGCTTATCAGA TCCTAATAC TCAACATCAG TCTGATAAGCTA 3 DB3 T10 N1 /5Phos/ GCTTATCAG AGCCTCAGCC TCAACATCAG TCTGATAAGCTACGGGCCTAGCAGCACGTA TCCTCCTTCCGCCAATATT
TACGTGCTGCTAGGCGCCTAGCTTATCAGA TCCTCCTTC TCAACATCAG TCTGATAAGCTAGGCCCGTA 4 DB3 T11 N1 /5Phos/GCTTATCAGCGCCTCAGCCTCAACATCAGTCTGATAAGCTACGGGCCTAGCAGCACGTCTCCTCCTTCCGCCAATATTTACGTGCTGCTAGGCGCCTAGCTTATCAGCTCCTCCTTCTCAACATCAGTCTGATAAGCTAGGCCCGTA 5 DB3 T12 N1 /5Phos/GCTTATCACCGCCTCAGCCTCAACATCAGTCTGATAAGCTACGGGCCTAGCAGCACGCCTCCTCCTTCCGCCAATATTTACGTGCTGCTAGGCGCCTAGCTTATCACCTCCTCCTTCTCAACATCAGTCTGATAAGCTAGGCCCGTA 6 DB3 T13 N1 /5Phos/GCTTATCCCCGCCTCAGCCTCAACATCAGTCTGATAAGCTACGGGCCTAGCAGCACCCCTCCTCCTTCCGCCAATATTTACGTGCTGCTAGGCGCCTAGCTTATCCCCTCCTCCTTCTCAACATCAGTCTGATAAGCTAGGCCCGTA 7 DB1 T10 N1 (Nt) /5Phos/ AAGCTA TTGTAATACCGACTCC TAGCTTATCAGA GCCCCC TCAACATCAG TCTGAT 8 DB3 T13 N1 (Nt) /5Phos/GCTTATCCCCGCCTCAGCCTCAACATCAGTCTGATAAGCTACGGGCCTAGCAGCACCCCTCCTCCTTCCGCCAATATTTACGTGCTGCTAGGCGCCTAGCTTATCCCCTCCTCCTTCTCAACATCAGTCTGATAAGCTAGGCCCGTA 9

The dumbbell-shaped nucleic acid template produced according to this example was confirmed through 15% dPAGE (denaturing PAGE) analysis. A 15% urea-polyacrylamide gel was produced using 7 M urea, 40% acrylamide/bis solution (29:1), 10% ammonium sulfate (APS), tetramethylethylenediamine (TEMED), and 1X TBE buffer (100 mM Tris, 90 mM boric acid, 1 mM EDTA). The linear template, ligated template, and exonuclease I/III-treated dumbbell template were denatured by heating in 1X TBE-urea sample buffer at 70°C for 5 minutes and then analyzed by 15% dPAGE in 0.5X TBE. After that, they were stained with 1X SYBR gold nucleic acid stain at 25°C for 30 minutes. The stained gel images were obtained through the ChemiDoc MP gel imaging system (Bio-Rad Laboratories) and processed using ImageJ software. The processed gel image results are as shown in Fig. 9. Referring to Fig. 9, it can be confirmed that the ligation was successful through the band positions, and ultimately, a dumbbell-shaped template that is not degraded by exonuclease was produced.

Confirmation of hybridization of the produced nucleic acid template with the target gene

Since the dumbbell-shaped nucleic acid template is designed to be able to complementarily bind to the target gene, hybridization with the target gene can occur. Hybridization of the produced nucleic acid template and the target gene was confirmed through 10% nPAGE (native PAGE). The dumbbell template (200 mM) and the target gene (200 mM) _{were reacted in 1X isothermal amplification reaction buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4 , 50} mM KCl, 2 mM _MgSO4 , 0.1% Tween 20, pH 8.8), and then annealed by slowly lowering the temperature from 80°C to 55°C. Afterwards, a 10% native polyacrylamide gel was prepared using 30% acrylamide/bis solution (29:1), 10% APS, TEMED, and 1X TBE buffer for nPAGE analysis. After analysis by 10% nPAGE in 0.5X TBE, the gel was stained with 1X SYBR gold nucleic acid stain for 30 minutes at 25℃. The stained gel image was obtained using a ChemiDoc MP gel imaging system, and the obtained gel image was processed using ImageJ software.

For each dumbbell template, hybridization with the target gene (miR-21) was analyzed through native PAGE, and the results are as shown in Fig. 10. Native PAGE is used to analyze whether the original secondary structure of the nucleic acid is formed, and denaturing PAGE is used to analyze the nucleic acid itself that has been thermally and chemically denatured. Therefore, whether the dumbbell template hybridizes with the target gene can be determined through the formation of a newly observed band in native PAGE.

As shown in Fig. 10, it was confirmed that hybridization with the target gene occurred in all dumbbell templates, and in particular, it was confirmed that hybridization with the target gene occurred better as the toehold region became longer in the dumbbell template (DB3 template) having a triple-arm. That is, the toehold region is preferably 10 to 30 bp.

Determination of the optimal temperature for RCA reaction using the fabricated triple-arm template

The optimal temperature for the RCA reaction using Bst 2.0 DNA polymerase was confirmed using the DB3 T13 N1 template that hybridized best with the target gene. To effectively induce the RCA reaction, the dumbbell template, target gene, and DNA polymerase were added at the very last step of preparing the RCA mater mix. The RCA mater mix was first prepared using MgSO ₄ (6 mM), dNTP (1 mM), and SYBR Green (2.5X) in 1X isothermal reaction buffer. After that, DB3 T13 N1 (200 nM) and Bst 2.0 DNA polymerase (0.08 U/μL) were added to prepare 18 μL of the mix, and 2 μL of the target gene (miR-21, 5 nM) was added to make a final volume of 20 μL. 2 μL of Tris-HCl buffer (20 mM) was used as a control. All reaction mixtures were prepared on ice, and the RCA reaction was performed at 50–65°C for 60 min and heated to 80°C for 20 min to inactivate Bst 2.0 DNA polymerase and terminate the reaction. Real-time fluorescence signals were measured at 1-minute intervals for 60 min using a QuantStudio ^TM 5 Real-Time PCR System (Applied Biosystems ^TM ). The net signal changes were obtained by subtracting the initial fluorescence measurement value (initial FI or FI ₀ ) from the fluorescence measurement value (fluorescence intensity, FI) of the corresponding sample at each time point. The relative fluorescence changes between the experimental groups were compared using the average fluorescence intensity (FI) obtained through 2–3 independent experimental repetitions, and the fluorescence signal changes according to the reaction conditions were analyzed using the values measured at 30 and 60 min.

In this example, in order to confirm the optimal temperature for the RCA reaction when using the template of the present invention, Bst 2.0 DNA polymerase was used, and the RCA reaction was performed using the template DB3 T13 N1 of the present invention at 50 to 65°C (inactivation temperature: 80°C). The basic reaction conditions are as shown in Table 4 below.

reagent density DB3 T13 N1 Template 200 nM Target gene 0 or 5 nM dNTP 1 mM MgSO ₄ 6 mM SYBR Green 2.5X Isothermal reaction buffer 1X Bst2.0 DNA polymerase 0.08 U/μL

The results according to this example are as shown in Fig. 11, and the results are the average of the results obtained by repeating the experiment 2-3 times (n). Referring to Fig. 11, it can be seen that the difference in fluorescence signal depending on the presence or absence of the target gene is most distinct when the RCA reaction temperature is 50°C, and therefore, it can be seen that the optimal reaction temperature in the RCA experiment using the DB3 template is about 40 to 60°C, preferably about 50°C.

Check RCA efficiency according to toehold length

The RCA efficiency according to the toehold region length was compared using dumbbell templates with different arms (DB1, DB2, DB3 templates). To effectively induce the RCA reaction, the dumbbell template, target gene, and DNA polymerase were added at the very last step of preparing the RCA mater mix. The RCA mater mix was first prepared using MgSO ₄ (6 mM), dNTP (1 mM), and SYBR Green (2.5X) in 1X isothermal reaction buffer. Then, the dumbbell template (200 nM) and Bst 2.0 DNA polymerase (0.08 U/μL) were added to prepare 18 μL of the mix, and 2 μL of the target gene (miR-21, 5 nM) was added to make a final volume of 20 μL. 2 μL of Tris-HCl buffer (20 mM) was used as a control. All reaction mixtures were prepared on ice, and the RCA reaction was performed at 50℃ for 60 min and heated at 80℃ for 20 min to inactivate Bst 2.0 DNA polymerase and terminate the reaction. Real-time fluorescence signals were measured at 1-minute intervals for 60 min using QuantStudio ^TM 5 Real-Time PCR System. The net signal changes were obtained by subtracting the initial fluorescence measurement value (FI ₀ ) from the fluorescence measurement value (FI) of the corresponding sample at each time point. The relative fluorescence changes between the experimental groups were compared using the average fluorescence intensity (FI) obtained through 2-3 independent experimental repetitions, and the fluorescence signal changes according to the reaction conditions were analyzed using the values measured at 30 and 60 min.

In this example, the template of the present invention was used to compare the RCA efficiency according to the length of the toehold portion, and the RCA reaction results at each length (10 to 13 bp) were confirmed. In this example, the reaction temperature was 50°C (deactivation temperature: 80°C). The specific reaction conditions in this example are as follows in Table 5.

reagent density Dumbbell Template 200 nM Target gene 0 or 5 nM dNTP 1 mM MgSO ₄ 6 mM SYBR Green 2.5X Isothermal reaction buffer 1X Bst2.0 DNA polymerase 0.08 U/μL

The results according to the present embodiment are as shown in Fig. 12, and the results are the average of the results obtained by repeating the experiment 2-3 times (n). Referring to Fig. 12, as the length of the toehold portion in the DB3 template increases, the RCA amplification efficiency increases. In particular, it can be confirmed that DB3 T13 N1 has a higher amplification efficiency than DB1 T10 N1 and DB2 T10 N1. That is, in the case of the DB3 template, when the length of the toehold portion is about 10 to 30 bp, preferably 13 bp, compared to the DB1 or DB2 template, the RCA amplification efficiency is further improved.

Comparison of RCA efficiency depending on whether the cleavage enzyme recognition site is included

To compare the RCA efficiency according to the inclusion of the cleavage recognition site in the dumbbell template, RCA was performed with dumbbell templates including the cleavage recognition site (Nt indicated) and dumbbell templates without the cleavage recognition site for DB1 and DB3 templates, respectively. To effectively induce the RCA reaction, the dumbbell template, target gene, and DNA polymerase were added at the very last step of preparing the RCA mater mix. The RCA mater mix was first prepared using MgSO ₄ (6 mM), dNTP (1 mM), and SYBR Green (2.5X) in 1X isothermal reaction buffer. Then, dumbbell template (200 nM) and Bst 2.0 DNA polymerase (0.08 U/μL) were added to prepare 18 μL of mix, and 2 μL of target gene (miR-21, 5 nM) was added to adjust the final volume to 20 μL. As a control, 2 μL of Tris-HCl buffer (20 mM) was used. All reaction mixtures were prepared on ice, and the RCA reaction was reacted at 50 °C for 60 min and heated at 80 °C for 20 min to inactivate Bst 2.0 DNA polymerase and terminate the reaction. Real-time fluorescence signals were measured at 1-minute intervals for 60 min using QuantStudio ^TM 5 Real-Time PCR System. The net signal changes were obtained by subtracting the initial fluorescence measurement value (FI ₀ ) from the fluorescence measurement value (FI) of the corresponding sample at each time point. The relative fluorescence changes between the experimental groups were compared using the average fluorescence intensity (FI) obtained through two independent experimental repetitions, and the fluorescence signal changes according to the reaction conditions were analyzed using the values measured at 30 and 60 min.

In order to compare the RCA efficiency according to the presence or absence of the cleavage enzyme recognition site in the dumbbell template of the present invention, RCA was performed with each template having the cleavage enzyme recognition site (indicated by Nt) and the template without the cleavage enzyme recognition site. In this example, the reaction temperature was 50°C (inactivation temperature: 80°C), and the specific reaction conditions in this example are as shown in Table 6 below.

reagent density Dumbbell Template 0.2 uM Target gene 0 or 5 nM dNTP 1 mM MgSO ₄ 6 mM SYBR Green 2.5X Isothermal reaction buffer 1X Bst2.0 DNA polymerase 0.08 U/μL

The results according to the present example are as shown in Fig. 13, and it can be confirmed that in the case of DB1 T10 N1, the Nt template (ver. 3) has lower fluorescence, and in the case of DB3 T13 N1, the Nt template has higher fluorescence. In other words, it can be seen that the Nt template (a template in which a cleavage enzyme recognition site exists) is more suitable for taking advantage of the triple-arm dumbbell template.

Confirmation of Nick RCA efficiency using cleavage enzyme

To compare the efficiency of Nick RCA using nicking enzyme and RCA without nicking enzyme, Nt template was used and Nt.BstNBI enzyme was added to RCA master mix. RCA master mix was prepared by adding MgSO ₄ (6 mM), dNTP (1 mM), SYBR Green (2.5X), and Bst 2.0 DNA polymerase (0.24 U/μL) to 1X isothermal reaction buffer. A mixture (18 μL) containing Nt.BstNBI (0.0375 U/μL) and nuclease-free water as a control was pre-incubated at 50°C for 3 min. The mixture and target gene (miR-21, 5 nM) were then added to make a final volume of 20 μL. 2 μL of Tris-HCl buffer (20 mM) was used as a control. All reaction mixtures were prepared on ice, and the RCA reaction was performed at 50°C for 60 min and heated at 80°C for 20 min to inactivate Bst 2.0 DNA polymerase and Nt.BstNBI cleavage enzyme and terminate the reaction. Real-time fluorescence signals were measured at 1-minute intervals for 60 min using QuantStudio ^TM 5 Real-Time PCR System. The net signal changes were obtained by subtracting the initial fluorescence measurement value (FI ₀ ) from the fluorescence measurement value (FI) of the corresponding sample at each time point. The relative fluorescence changes between the experimental groups were compared using the average fluorescence intensity (FI) obtained through two independent experimental repetitions.

In this example, the detection efficiency of the target substance was confirmed depending on the presence or absence of the cleavage enzyme. In this example, the reaction temperature was 50°C (deactivation temperature: 80°C), and the specific reaction conditions in this example are as shown in Table 7 below.

reagent density Dumbbell Template 200 nM Target gene 0 or 5 nM dNTP 0.32 mM MgSO ₄ 6 mM SYBR Green 2.5X Isothermal reaction buffer 1X Bst2.0 DNA polymerase 0.24 U/μL Nt.BstNBI cleavage enzyme 0.0375 U/μL

The results according to this example are as shown in Fig. 14, and it can be confirmed that when a Nick RCA reaction is induced using a nicking enzyme, the fluorescence increases exponentially compared to the existing RCA. However, in the case of the DB1 T10 N1 (Nt) template, the non-specific reaction also increased exponentially. In contrast, in the case of the DB3 T13 N1 (Nt) template, the signal increases rapidly due to the target gene, and the non-specific reaction is relatively less compared to DB1. It can be confirmed that the existing RCA also shows higher RCA efficiency than DB1, similar to the previous results.

That is, according to the present embodiment, when the template (DB3) of the present invention having a three-arm structure is used, it can be seen that the RCA efficiency is maximized and non-specific reactions are reduced depending on the presence of the cleavage enzyme, thereby enabling specific detection of the target substance.

Confirmation of Nick RCA efficiency using cleavage enzyme

In this example, RCA and Nick RCA were performed on each DB1 and DB3 template depending on the presence or absence of the target gene or the presence or absence of a nicking enzyme, and 10% native PAGE analysis was used to analyze each obtained DNA product.

As can be seen in the gel image of Fig. 15, it can be seen that nicking of the DNA strand occurred effectively in the DNA product of Nick RCA (confirmation of DNA fragments of various sizes), and in the case of DB1, it can be seen that Nick RCA occurred even in the absence of the target gene (non-target). On the other hand, in the case of DB3, it can be seen that there is a clear difference in the amount of DNA product depending on the presence or absence of the target gene in the Nick RCA reaction compared to DB1. That is, it can be confirmed that the detection efficiency of the target material is high when the template (DB3) of the present invention having the three-arm structure of the present invention is used.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Attach electronic file of sequence list

Claims (14)

A nucleic acid template comprising at least one genetic sequence of an arm structure,
The gene sequence of the above arm structure consists of a stem sequence and a loop sequence,
A dumbbell-shaped rolling circle amplification reaction nucleic acid template, wherein a toehold site and a cleavage enzyme recognition site are located on the gene sequence of the above-mentioned arm structure. In the first paragraph,
A nucleic acid template, wherein the above-mentioned cleavage enzyme recognition site is located on a loop sequence. In the first paragraph,
The above cleavage enzyme recognition site is
A nucleic acid template, wherein the nucleic acid template is located on a sequence located 1 to 15 nt away from a toehold site, or on a sequence of a loop in which a toehold site is not located. In the first paragraph,
The above template is a nucleic acid template having a length of 100 to 150 nt. In the first paragraph,
A nucleic acid template, wherein the stem sequence has a length of 10 to 30 bp. In the first paragraph,
A nucleic acid template, wherein the above loop sequence has a length of 15 to 45 bp. In the first paragraph,
A nucleic acid template comprising two or three arm structural gene sequences. In the first paragraph,
The above-mentioned toehold region is a nucleic acid template having a length of 10 to 30 bp. In the first paragraph,
A nucleic acid template, wherein the above cleavage enzyme recognition site has a length of 10 to 30 bp. In the first paragraph,
The above template is a nucleic acid template consisting of any one of the gene sequences represented by SEQ ID NOs: 2 to 9. A composition for a rolling circle amplification reaction comprising a nucleic acid template of any one of claims 1 to 10. A method for detecting a target gene using a nucleic acid template of the first clause,
a) a step of introducing a sample into a composition comprising a nucleic acid template, a primer and a cleavage enzyme of the first clause; and
b) A method for detecting a target gene, comprising the step of performing a rolling circle amplification reaction. In Article 12,
The step b) above is a method for detecting a target gene, wherein, if a target gene is present in a sample, i) a step in which the target gene and a primer bind to a nucleic acid template; and ii) a step in which a gene polymerase binds, the dumbbell-shaped nucleic acid template is released, and a roll-over amplification reaction proceeds. In Article 12,
A method for detecting a target gene, comprising: after step b), a step c) of adding a fluorescent substance capable of binding to the product according to step b), and determining that the target substance exists when fluorescence appears.