KR102529424B1 - DNA structure for detection of target nucleic acid, composition for nucleic acid detection, and nucleic acid detection method using the same - Google Patents

Abstract

The present invention relates to a DNA construct for detecting a target nucleic acid, a composition for detecting a nucleic acid comprising the DNA construct, and a method for detecting a nucleic acid using the same, and more particularly, the DNA construct has a hairpin structure, and the DNA construct It relates to a composition for detecting nucleic acids and a method for detecting nucleic acids for simple and rapid colorimetric detection of target nucleic acids through the detection of pyrophosphate as a medium.

Description

DNA structure for detection of target nucleic acid, composition for nucleic acid detection, and nucleic acid detection method using the same

The present invention relates to a DNA construct for detecting a target nucleic acid, a composition for detecting a nucleic acid comprising the DNA construct, and a method for detecting a nucleic acid using the same, and more particularly, the DNA construct has a hairpin structure, and the DNA construct The present invention relates to a composition for detecting nucleic acids and a method for detecting nucleic acids for simple and rapid colorimetric detection of target nucleic acids through detection of pyrophosphate (PPi) through the medium.

miRNAs are single-stranded, small, non-coding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression, and are biomarkers of human diseases, including cancer, caused by their dysregulation. is used to diagnose Detection of miRNA requires very high selectivity because the base sequences of miRNA families are similar, and high sensitivity because the amount of miRNA is as small as 0.2 fM to 20 pM.

Traditional detection methods for miRNA include RT-PCR, northern blot, cloning and microarray. Each method had problems such as complicated process, time-consuming operation, low selectivity and sensitivity, and the need for expensive detection equipment. Although qPCR is among the most common miRNA detection methods, an efficient method for miRNA detection is difficult because it involves complex steps such as extraction and isolation of miRNA from blood for diagnosis, and elongation and transcription of miRNA for PCR. no. In addition, since the length of RNA is about 19 to 23 nucleotides, there is a problem that it is too short for gene amplification using PCR.

Recently, various advanced miRNA detection methods based on gene amplification such as RCA (Rolling Circle Amplification) and HCR (Hybridization Chain Reaction) have been developed. Other approaches based on signal amplification are also being developed, such as FRET, electrochemical analysis, and enzymatic signal amplification.

On the other hand, as a method of diagnosing genes for on-site diagnosis, several oligonucleotides that have undergone a complex synthesis process and a method of amplifying signals with fluorescence labeled on genes have been used. However, these methods are time consuming and costly, and because they require a fluorescence analyzer, they are difficult to use for point-of-care diagnostics that require simple and rapid diagnosis.

In order to solve these problems, a colorimetric detection method that is easily accessible to users and does not require an analysis device to read the detection result is being developed. However, most colorimetric methods have limitations in detection, lack of sensitivity, and require time-consuming work due to specific labeling and complicated procedures. Therefore, it is required to develop a colorimetric detection method for point-of-care treatment that has high sensitivity and selectivity and enables rapid detection.

C. Wang et al., Analytica Chimmica Acta, 987, 111-117, 2017 B.S. Batule et al., Journal of Agricultural and Food Chemistry. 66, 3003-3008, 2018

An object of the present invention is to provide a composition for detecting nucleic acids and a method for detecting nucleic acids for simple and rapid colorimetric detection of target nucleic acids through the detection of pyrophosphate (PPi) through a DNA structure having a hairpin structure. .

In order to achieve the above object, the present invention sequentially from the 5' direction to the 3' direction,

1) primer hybridization domain sequences;

2) target nucleic acid hybridization domain sequence;

3) primer extension domain sequences; and

4) Antisense nucleotide sequence complementary to the sequence of 1) above

made up of,

The sequence of 2) and the sequence of 3) provide a DNA structure in which all or part forms a single hairpin structure.

In one embodiment of the present invention, the target nucleic acid may be microRNA (miRNA), and more specifically, the miRNA may be any one selected from miRNA21, miRNA24-3P, miRNA25b, and miRNA146a, but is not limited thereto. .

In addition, the present invention provides a composition for detecting nucleic acids comprising the DNA construct.

In another embodiment of the present invention, the nucleic acid may be miRNA, and more specifically, the miRNA may be any one selected from miRNA21, miRNA24-3P, miRNA25b, and miRNA146a, but is not limited thereto.

In another embodiment of the present invention, the detection may be detection of pyrophosphate (PPi).

In another embodiment of the present invention, the composition may further include any one or more selected from nPfu special DNA polymerase and Duplex Specific Nuclease (DSN).

In addition, the present invention comprises (a) mixing the DNA construct and the nucleic acid;

(b) mixing the nPfu special enzyme and DSN with the reactants mixed in step (a); and

(c) adding a compound represented by the following [Formula 1] to the reaction product generated in step (b);

[Formula 1]

In another embodiment of the present invention, the nucleic acid may be miRNA, and more specifically, the miRNA may be any one selected from miRNA21, miRNA24-3P, miRNA25b, and miRNA146a, but is not limited thereto.

In another embodiment of the present invention, pyrophosphate may be released after the nPfu special enzyme and DSN are mixed in step (b).

In another embodiment of the present invention, the released pyrophosphoric acid may change the color of the compound represented by Chemical Formula 1 to colorless.

The DNA construct according to the present invention, the composition for detecting nucleic acid containing the construct, and the method for detecting nucleic acid have high sensitivity and selectivity, and miRNA diagnosis is possible simply and quickly, thereby reducing time and effort and color change. Since it can be detected with a low temperature, it can be used for point-of-care

In addition, since it can be cleaved by Duplex Specific Nuclease (DSN), which can cleave DNA from RNA-DNA hybrids, and the target miRNA can be recycled and signal amplification is possible, diagnosis can be made even with a very small amount of miRNA. there is

Figure 1 shows the oligonucleotide sequences used in the present invention, in SEQ ID NO: 2 to SEQ ID NO: 5, orange represents the target nucleic acid hybridization domain, blue represents the primer extension domain, green represents the primer hybridization domain , the underline indicates the stem of the hairpin structure.
2 is a schematic diagram of a structure of a DNA construct according to the present invention and a nucleic acid detection process using the DNA construct.
Figure 3 is PAGE denaturing result data to confirm deblocking of the hairpin probe (h-Probe) in the presence of a matching target miRNA, (a) is h-Tem 21, a hairpin template for miRNA21 detection h-Probe with (h-Probe 21), (b) h-Probe with h-Tem 24-3P (h-Probe 24-3P) for detecting miRNA24-3P, (c) for detecting miRNA25b h-Probe with h-Tem 25b (h-Probe 25b), (d) is the result of using the h-Probe with h-Tem 146a (h-Probe 146a) for detecting miRNA146a. wherein lane 1 contains h-Pri, lane 2 contains h-Tem, lane 3 contains h-Pri and h-Tem, lane 4 contains h-Pri, h-Tem and nPfu special enzymes Lane 5 contains h-Pri, h-Tem, nPfu special enzyme and miRNA21, lane 6 contains h-Pri, h-Tem, nPfu special enzyme and A24-3P, and lane 7 contains h-Pri, h-Tem, nPfu special enzyme and A24-3P. -Pri, h-Tem, nPfu special enzyme and miRNA25b, lane 8 contains h-Pri, h-Tem, nPfu special enzyme and miRNA146a.
Figure 4 shows the amplification and selectivity study results of the h-Probe using the nPfu special enzyme and DSN, (a) is the denaturing PAGE data for the selectivity of the corresponding h-Probe with matching and inconsistent miRNAs, and lane 1 is h -Probe, lane 2 contains h-Probe and nPfu special enzyme, lane 3 contains h-Probe, nPfu special enzyme and miRNA21, lane 4 contains h-Probe, nPfu special enzyme and miRNA24-3P Lane 5 contains h-Probe, nPfu special enzyme and miRNA25b, Lane 6 contains h-Probe, nPfu special enzyme and miRNA146a. (b) is a colorimetric detection result using a pp probe after the reaction.
Figure 5 shows the results of the sensitivity study of h-Probe using nPfu special enzyme and Duplex Specific Nuclease (DSN), (a) is for detecting four miRNAs by concentration (1 nM to 1 fM) including negative control. It shows the LoD result of colorimetric analysis, (b) is the absorbance spectrum result for the sensitivity of absorbance analysis depending on the concentration (10 pM to 10 aM) of miRNA25b, and (c) is the absorbance response (λab = 560 nm) and the target The linear relationship between the logarithm of miRNA25b concentration is graphed (Fo=absorption intensity of no target and F=absorption intensity of miRNA25b addition).
Figure 6 shows the results of high sensitivity studies of h-Probe using nPfu special enzyme and DSN for colorimetric analysis by pp probe when the reaction time is increased to 1 hour.
Figure 7 (a) is the fluorescence spectrum result (excitation at 400 nm) for the sensitivity of fluorescence analysis depending on the concentration (10 pM to 1 aM) of miRNA25b, and (b) is the fluorescence response (λem = 464 nm) and the target The linear relationship between the log of miRNA25b concentration is graphed (Fo = fluorescence intensity of no target and F = fluorescence intensity with miRNA25b addition).

Hereinafter, the present invention will be described in detail.

The present invention sequentially from the 5' direction to the 3' direction, 1) the primer hybridization domain base sequence; 2) target nucleic acid hybridization domain sequence; 3) primer extension domain sequences; and 4) an antisense nucleotide sequence complementary to the sequence of 1), wherein the sequence of 2) and the sequence of 3) form a single hairpin structure in whole or in part.

According to one embodiment of the present invention, the target nucleic acid may be miRNA, and more specifically, the miRNA may be any one selected from miRNA21, miRNA24-3P, miRNA25b, and miRNA146a, but is not limited thereto.

In the present invention, a hairpin template (h-Tem) including three domains, the primer hybridization domain, the target nucleic acid hybridization domain, and the primer extension domain, was designed to detect the target nucleic acid via the hairpin structure. Here, a hairpin probe (h-probe) was prepared by annealing the primer capable of hybridizing to the h-tem (SEQ ID NO: 1, 5'-CATGCATGCATGCAT-3' in FIG. 1).

The sequences of the miRNAs miRNA21, miRNA24-3P, miRNA25b and miRNA146a can be represented by SEQ ID NO: 6 to SEQ ID NO: 9 in FIG. 1, respectively. -probe sequence h-probe 21 (SEQ ID NO: 2, 5'-TCAACATCAGTCTGATAAGCTAGCTAGCTACCATGTTGAATGCATGCATGCATG-3'), h-probe 24-3P (SEQ ID NO: 3,5'-CTGTTCCTGCTGAACTGAGCCAGCTAGCTACGGGAACAGATGCATGCATGCATG-3'), h-probe 25b (SEQ ID NO: 4 5'-TCACAAGTTAGGGTCTCAGGGTGCTAGCTACGCTTGTGAATGCATGCATGCATG-3'), h-probe 146a (SEQ ID NO: 5, 5'-AACCCATGGAATTCAGTTCTCAGCTAGCTACGATGGGTTATGCATGCATGCATG-3') was prepared.

2, the DNA structure according to the present invention is composed of a primer hybridization domain (green), a target nucleic acid hybridization domain (orange), and a primer extension domain (blue), forming a hairpin structure. Here, by inserting a sequence complementary to the target nucleic acid at the end of the hairpin-shaped DNA structure, the hairpin structure can be released to form straight DNA when a matching target is present.

In addition, the present invention provides a composition for detecting nucleic acids comprising the DNA construct.

According to another embodiment of the present invention, the nucleic acid may be miRNA, and more specifically, the miRNA may be any one selected from miRNA21, miRNA24-3P, miRNA25b, and miRNA146a, but is not limited thereto.

Also, according to another embodiment of the present invention, the detection may be detection of pyrophosphate.

In addition, according to another embodiment of the present invention, the composition may further include any one or more selected from nPfu special DNA polymerase and Duplex Specific Nuclease (DSN).

If a primer is attached to the end of the hairpin DNA to make a detection substance (h-Probe), the h-Probe does not undergo polymerization by the nPfu Special enzyme. However, when the hairpin structure is released due to the presence of the target material, the reaction between the primer and the enzyme becomes operable, and pyrophosphate can be produced as a by-product through the polymerization reaction (FIG. 2).

The attached RNA can reuse the RNA by using the property of selectively cutting only the DNA strand in the DNA-RNA duplex structure, which is a characteristic of the DSN, so that the signal can be continuously amplified with a small amount of RNA. RNA reused in this way can be attached to other h-Probes, continuously enabling DNA polymerization, and producing dozens of pyrophosphates per hairpin DNA structure (FIG. 2).

The present invention comprises the steps of (a) mixing the DNA construct of claim 1 and the nucleic acid; (b) mixing the nPfu special enzyme and DSN with the reactants mixed in step (a); and (c) adding a compound represented by Formula 1 to the reaction product generated in step (b).

[Formula 1]

According to another embodiment of the present invention, the nucleic acid may be miRNA, and more specifically, the miRNA may be any one selected from miRNA21, miRNA24-3P, miRNA25b, and miRNA146a, but is not limited thereto. .

In addition, according to another embodiment of the present invention, pyrophosphate may be released after the nPfu special enzyme and DSN are mixed in step (b).

In addition, according to another embodiment of the present invention, the released pyrophosphoric acid can change the color of the compound represented by Chemical Formula 1 to colorless.

Compound 1 has a Cu ²⁺ chelate structure, and reacts with pyrophosphate to form a pyrophosphate-copper complex, change color from pink to colorless, and emit fluorescence. Through this color change, it is possible to quickly diagnose nucleic acids in the field.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Preparation of h-probe

Lyophilized oligonucleotides (Cosmogenetech, Korea) were diluted in 25 mM Trizma buffer (50 mM NaCl and 10 mM MgCl ₂ ) to a concentration of 0.1 mM. h-Pri and h-Tem were mixed at equal concentrations, heated to 95˚C and cooled to 4˚C for annealing.

Example 2. Confirmation of h-probe deblocking by miRNA

10 μM h-Probe and 10 μM target miRNA were added to the reaction tube. To the reaction, 1.5 μL of 10 x nPfu special enzyme buffer (200 mM Tris-HCl / pH 8.8, 500 mM KCl, 25 mM MgCl ₂ ) and 5 μL of each 2 mM dNTP mixture (dATP, dCTP, dGTP and dTTP) were added, followed by water. Fill up to 14 μL solution. 1 μL of 2 units of nPfu special enzyme (Enzynomics, Korea) was added to the reaction, and the reaction was incubated at 37 ° C for 30 minutes. The product solution was mixed with 1.7 μL of 10 x loading buffer and loaded under 20% denaturing PAGE.

Blocking of primer extension by the nPfu special enzyme due to the hairpin structure of the DNA construct of the present invention and formation of a miRNA and template duplex from the hairpin structure in the presence of the target miRNA were tried to be demonstrated. As a result, as shown in Figure 3, lane 4 demonstrated that primer extension using the nPfu special enzyme in the hairpin template (h-tem) did not work. However, Lanes 5 to 8 showed that each h-Probe could function for primer extension in the presence of each matching target miRNA. All primer extensions were stopped at the starting positions of the miRNA and template duplex, and the corresponding 32mer primer extension products appeared. However, primer extension products were not observed for mismatched target miRNAs. Therefore, through this result, it was confirmed that the present invention using the nPfu specific enzyme is capable of detecting miRNA through a DNA structure.

Example 3. Confirmation of amplification and selectivity of h-probe using nPfu special enzyme and DSN

10 μM of h-Probe and 1 nM of target miRNA were added to the reaction tube. To the reaction, 1.5 μL of 10 × nPfu special enzyme buffer (200 mM Tris-HCl/pH 8.8, 500 mM KCl, 25 mM MgCl ₂ ) and 5 μL of each 2 mM dNTP mixture (dATP, dCTP, dGTP and dTTP) were added. Next, fill up with water to make a 13 μL solution. 2 units of nPfu special enzyme in 1 μL and 1 unit of DSN in 1 uL were added to the reaction mixture. Reactions were incubated at 37 °C for 30 min. The product solution was mixed with 1.7 μL of 10 x loading buffer and loaded under 20% denaturing PAGE.

For colorimetric detection, 1 μL of 25 mM pp probe represented by Chemical Formula 1 was added to the final reactant.

The nPfu special enzyme requires a high concentration of miRNA for sufficient primer extension, and a sensitive colorimetric signal cannot be obtained in a reaction containing only a very small amount (pM to fM) of the target miRNA. In order to improve this sensitivity problem, a signal amplification method using a DSN enzyme that cleaves the DNA template in a duplex of miRNA and DNA template was selected. In this process, miRNAs were released and were expected to be recycled for primer extension.

As shown in Figure 4a, 32mer primer extension products corresponding to each target miRNA (miRNA21, miRNA24-3P, miRNA25b, and miRNA146a) were observed, but 32mer primer extension products could not be observed with other untargeted miRNAs. This shows that the hairpin-mediated miRNA detection system has amplification and selectivity.

In addition, expecting that the pyrophosphate generated during the primer extension process would react with the pp probe, the pp probe was added to this solution, and the pyrophosphate removed copper divalent ions from the pp probe and bound them to form a pyrophosphate-copper complex. By doing so, a color change from pink to colorless was observed (Fig. 4b).

Example 4. Sensitivity confirmation of h-probe using nPfu special enzyme and DSN

The experimental method was the same as in Example 3, and target miRNAs at different concentrations (1 fm to 1 nM) were used.

For colorimetric detection, 1 μL of 25 mM pp probe was added to the final reactant, and accurate LoD was measured based on the UV graph from the colorimetric detection result.

Using the signal amplification system using the DSN enzyme, we investigated whether the sensitivity of the h-Probe system varies depending on the concentration of miRNA (1nM-1fM). As shown in FIG. 5a, it was observed that the color of the target miRNA rapidly changed from pink to colorless with a very small amount, indicating that the signal amplification system using the DSN enzyme was working.

In addition, we observed slight differences in the sensitivity of color change depending on the characteristics of the target miRNA. The sensitivity of the probes for miRNA25b and miRNA24-3P appeared to be slightly higher compared to miRNA21 and miRNA146a.

The probe for miRNA25b was found to have the highest sensitivity when visually confirmed, and the absorption spectrum was measured while changing the concentration of miRNA25b from 10 pM to 10 aM (FIG. 5b). To investigate the exact limit of detection (LoD), we based UV absorption data with concentrations of various target miRNAs, including miRNA25b (Fig. 5c). The 3σ method was used [LoD = 3×(SD/S), where SD is the standard deviation and S is the slope of the logarithmic plot].

As shown in Fig. 5c, miRNA25b showed a very low LoD of 31.6aM, and other target miRNAs also showed a low LoD ranging from 2.1fM to 31.6aM. In conclusion, the LoDs of miRNA25b and miRNA24-3P were 31aM and 703aM, respectively, confirming that they were more sensitive than miRNA21 and miRNA146a (LoD = 1.9fM, 2.1fM).

In addition, since the reaction rate differs depending on the GC content of miRNA, it is expected that the sensitivity will increase as the reaction time increases, and when the detection time is increased from 30 minutes to 1 hour, it is possible to detect miRNA at a much lower concentration with the naked eye. confirmed (FIG. 6).

Therefore, h-Probe equipped with a signal amplification system is judged as a detection method with sufficient sensitivity because the concentration of miRNA in cancer cells is about 0.2 fM to 20 pM.

In addition, the pp probe exhibited fluorescence and was expected to be usable for more sensitive detection, and sensitivity was investigated using fluorescence including target miRNA25b, which is the most sensitive miRNA (FIG. 7a). As a result, the fluorescence detection sensitivity for miRNA25b was determined using the 3σ method, and LoD was 41.9aM, confirming that it was similar to the colorimetric method (FIG. 7b).

In the present invention, colorimetric detection of a target miRNA with a low detection limit (LoD = 10 fM) using a pp probe took only 30 minutes, and it was confirmed that it was selective. Therefore, when the composition for detecting nucleic acid and the method for detecting nucleic acid of the present invention are used, detection is possible by color change, detection time is reduced, and sensitivity is high, so that it can be used for on-site diagnosis.

As described above, the present invention has been described through examples. Those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. Therefore, the experimental examples described above are illustrative in all respects and should be understood as non-limiting ones. The scope of the present invention is indicated by the claims to be described later rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be construed as being included in the scope of the present invention.

<110> INDUSTRIAL COOPERATION FOUNDATION JEONBUK NATIONAL UNIVERSITY <120> DNA structure for detection of target nucleic acid, composition for nucleic acid detection, and nucleic acid detection method using the same <130> P-2021-002 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 15 <212> DNA <213> artificial sequence <220> <223> h-Pri <400> 1 catgcatgca tgcat 15 <210> 2 <211> 54 <212> DNA <213> artificial sequence <220> <223> h-Tem 21 <400> 2 tcaacatcag tctgataagc tagctagcta ccatgttgaa tgcatgcatg catg 54 <210> 3 <211> 54 <212> DNA <213> artificial sequence <220> <223> h-Tem 24-3P <400> 3 ctgttcctgc tgaactgagc cagctagcta cgggaacaga tgcatgcatg catg 54 <210> 4 <211> 54 <212> DNA <213> artificial sequence <220> <223> h-Tem 25b <400> 4 tcacaagtta gggtctcagg gtgctagcta cgcttgtgaa tgcatgcatg catg 54 <210> 5 <211> 54 <212> DNA <213> artificial sequence <220> <223> h-Tem 146a <400> 5 aacccatgga attcagttct cagctagcta cgatgggtta tgcatgcatg catg 54 <210> 6 <211> 22 <212> RNA <213> artificial sequence <220> <223> miRNA21 <400> 6 uagcuuauca gacugauguu ga 22 <210> 7 <211> 22 <212> RNA <213> artificial sequence <220> <223> miRNA24-3P <400> 7 uggcucaguu cagcaggaac ag 22 <210> 8 <211> 22 <212> RNA <213> artificial sequence <220> <223> miRNA25b <400> 8 cccuugagac ccuaacuugu ga 22 <210> 9 <211> 22 <212> RNA <213> artificial sequence <220> <223> miRNA146a <400> 9 ugagaacuga auuccaugg uu 22

Claims (13)

delete delete delete delete delete delete delete delete (a) mixing the DNA construct and the nucleic acid;
(b) mixing nPfu special enzyme (nPfu special DNA polymerase) and DSN (Duplex Specific Nuclease) with the reactants mixed in step (a); and
(c) In the nucleic acid detection method comprising the step of adding a compound represented by [Formula 1] to the reactant generated in step (b),
In step (b), pyrophosphate is released after the nPfu special enzyme and DSN are mixed, and the released pyrophosphate changes the color of the compound represented by Formula 1 to colorless,
The DNA structure,
Sequentially from the 5´ direction to the 3´ direction,
1) target nucleic acid hybridization domain sequence;
2) primer extension domain sequences; and
3) consists of a primer hybridization domain nucleotide sequence,
The primer hybridization domain nucleotide sequence is hybridized with the antisense nucleotide sequence of SEQ ID NO: 1,
The sequence of 1) and the sequence of 2) form a single hairpin structure in whole or in part,
Wherein the nucleic acid is any one nucleic acid selected from the group consisting of miRNA21, miRNA24-3P, miRNA25b and miRNA146a.
[Formula 1]

.
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