KR102375106B1 - System for detecting nucleic acid - Google Patents

Abstract

The present invention relates to a method for detecting and amplifying a target nucleic acid by converting it into a universal code having a unified specific sequence. Since a nucleic acid can be converted/unified into a pre-designated universal code, it is not necessary to design a new probe for each target nucleic acid every time, which can be usefully applied to a detection system having a complex structure.

Description

Nucleic acid detection system {SYSTEM FOR DETECTING NUCLEIC ACID}

The present invention relates to detecting and amplifying a target nucleic acid by converting it into a unified universal code.

A method for detecting a specific nucleic acid (DNA or RNA) or protein is a fundamentally important technique in the field of scientific research. Being able to detect and identify specific nucleic acids or proteins allows researchers to determine which genetic or biological markers are indicative of a person's health status. By using such a method for detecting nucleic acids and proteins, it is possible to detect a modification of a pathogen gene or expression of a specific gene present in a sample. Such molecular diagnosis is used to diagnose the root of diseases such as DNA or RNA, and is used in various fields such as infectious diseases, cancer diagnosis, genetic diseases, and customized diagnosis. A representative molecular diagnostic technique includes a PCR technique that amplifies DNA within a short time (Saiki, R., et. al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-91. 1998). However, the general PCR technique must use the electrophoresis method to confirm the amplified DNA. In order to perform such electrophoresis, there has been troublesome to make an agarose gel and to confirm the DNA by staining it with EtBr or the like. In addition, since the recently used real-time PCR method uses fluorescence, electrophoresis is unnecessary, but expensive equipment and expensive fluorescent reagents must be used (Higuchi, R., et. al., Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions (Nature Biotechnology 11, 1026 - 1030, 1993) has a problem. Recently, Cepheid's GeneXprt system and reagents, a PCR product with a point-of-care concept, have been developed and sold, but since the equipment and reagents are very expensive, it is difficult to use them in general tests (Helb, D., et. al., Rapid Detection). of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand, Near-Patient Technology. J. Clin. Microbiol. 48, 229-237, 2010). Alternatively, there is the Nucleic Acid Lateral Flow Assay, which uses a membrane instead of gel electrophoresis after PCR to confirm (Aveyard, J., et. al., One step visual detection of PCR products). with gold nanoparticles and a nucleic acid lateral flow (NALF) device. Chem. Commun., 41, 4251-4253, 2007). However, compared to gel electrophoresis technology, it is difficult to manufacture and use in a laboratory because it is more complicated, and the probe sequence attached to the membrane is universally used because of the limitation of the technology that must be used to specifically bind according to the PCR amplification product. There are limitations to its use. In particular, this PCR-based smoke sequencing method is extremely vulnerable to specific errors and there are frequent serious misdiagnosis due to problems in the data interpretation process. Although studies are underway to express color change, etc., most of these studies detect only one biomarker at a time, so it is difficult to effectively diagnose most diseases involving multiple biomarkers.

Meanwhile, as a popular cancer diagnosis technique, various methods such as molecular biological diagnosis, ultrasound diagnosis, X-ray, MRI, CT, PET, and bone scan exist. Since most cancer diagnosis techniques are ultimately judged by the naked eye, a large error may occur in the radiographic film or tissue reading process. That is, depending on the size of the cancer, it may not be detected or a simple inflammation may be mistakenly diagnosed as cancer. Some methods such as MRI and PET require a long measurement time, but there is a disadvantage that the patient's movement and tension affect the accuracy of diagnosis. In addition, the risk of radiation exposure to the patient is an unavoidable problem. Therefore, for a more reliable cancer diagnosis, tumor tissue is directly collected and tested through biopsy. . The use of needles or surgical therapy to extract a tumor can also cause great objection to the patient, and there are cases where tissue biopsy itself is not possible depending on the patient's condition. In general, existing methods used to confirm cancer are expensive and inconvenient to patients, so they are not suitable for periodic and frequent examinations. Currently, attempts are gradually being made to diagnose cancer by detecting biomarkers therein through body fluids such as blood, feces, and saliva through a liquid biopsy. Various studies have already revealed that specific nucleotide sequences exist in large numbers in the blood of cancer patients compared to those who are not patients, and all data is stored in the form of bio-big data. As for the genetic biomarkers currently being studied, small non-coding RNAs (sncRNAs) such as miRNAs are attracting attention. Recently, it has been known that RNA and DNA interact with each other to greatly affect cell division, growth, death, and tumor progression. In addition, although studies on the correlation between sncRNA profiling and genetic variation of genes are being conducted, there has been a problem in that each probe needs to be designed according to various short sequences that are noticed in sequencing analysis. In addition, since human diseases are more often caused by problems with multiple genes rather than a single gene abnormality, the accuracy and usefulness of diagnosis can be improved if multiple genes can be identified in a single test.

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

It is an object of the present invention to provide a nucleic acid detection system.

It is also an object of the present invention to provide a nucleic acid detection signal amplification system.

It is also an object of the present invention to provide a universal code conversion system.

It is also an object of the present invention to provide a method for detecting a nucleic acid or amplifying a detection signal.

In addition, it is an object of the present invention to provide a biosensor for detecting nucleic acids.

In order to solve the above problems, the present invention provides a nucleic acid detection system comprising a first nucleic acid S1, a second nucleic acid O, a third nucleic acid F1, a fourth nucleic acid S2, a fifth nucleic acid U, and a sixth nucleic acid F2.

In addition, the present invention provides a nucleic acid detection signal amplification system comprising a first nucleic acid S1, a second nucleic acid O, a third nucleic acid F1, a fourth nucleic acid S2, a fifth nucleic acid U, and a sixth nucleic acid F2.

The present invention also provides a universal code conversion system comprising a first nucleic acid S1, a second nucleic acid O, a third nucleic acid F1, a fourth nucleic acid S2, a fifth nucleic acid U and a sixth nucleic acid F2.

In addition, the present invention provides a method for detecting a nucleic acid or amplifying a detection signal through a toehold-mediated strand displacement reaction.

In addition, the present invention provides a biosensor for detecting nucleic acids comprising a hybrid of a third nucleic acid F1 and a sixth nucleic acid F2, S10 and S2U.

According to the present invention, a target nucleic acid can be detected or a detection signal can be amplified by a null reaction, and various target nucleic acids can be converted/unified into a pre-specified universal code, so that a probe for each target nucleic acid can be newly designed every time. Since there is no need, it can be usefully applied to a detection system having a complex structure.

1 is a schematic diagram showing the sequence conversion process through the toehold-mediated strand displacement reaction of the system of the present invention:
Universal code: fifth nucleic acid U;
Substrate 1: first nucleic acid S1;
Output: second nucleic acid O;
miRNA: target nucleic acid (T);
Fuel 1: third nucleic acid F1;
Substrate 2: fourth nucleic acid S2; and
Fuel 2: Sixth Nucleic Acid F2.
2 is a diagram comparing the thermodynamic energies of the core constituent materials in each step constituting the system of the present invention.
3 is an electrophoresis result confirming the reaction progress in Cycle 1 of the system of the present invention according to changes in reaction temperature and reaction time under experimental conditions.
4 is a diagram confirming the sequence optimization of nucleic acids constituting the system of the present invention by electrophoresis.
5 is a result of confirming the reaction progress of each step of cycle 1 and cycle 2 by the electrophoresis method in the process of converting the target nucleic acid to the universal code (U) in the system of the present invention.
6 is a result confirming the reaction progress of the universal code according to the concentration of the target nucleic acid.
7 is a diagram confirming the results of amplification and detection of the system of the present invention according to the presence or absence of 12 miRNAs through fluorescence kinetic analysis. 7a is a diagram confirming the fluorescence detection results for 12 types of miRNAs with a universal code for a specific target miRNA (miR-21). 7B is a diagram illustrating the conversion of each target miRNA into a universal code using the system of the present invention, and confirming the result of fluorescence detection for each target miRNA.
8 is a diagram showing the electrophoresis results regarding the reaction progress of the cycle 1 part in the system of the present invention for 12 miRNAs.
9 is a diagram showing the electrophoresis results for the reaction progress of the cycle 2 part in the system of the present invention for 12 miRNAs.
10 shows the application of the nucleic acid detection system of the present invention to a graphene biosensor. The left side shows the experimental outline of Example 5, and the right side shows the measured fluorescence values.
11 is a schematic diagram showing the sequence complementarity of a first nucleic acid S1, a second nucleic acid O, a third nucleic acid F1, a fourth nucleic acid S2, a fifth nucleic acid U, and a sixth nucleic acid F2 constituting the system of the present invention:
*: sequence complementary.

Hereinafter, the present invention will be described in detail by way of embodiments of the present invention. However, the following embodiments are presented as examples for the present invention, and the present invention is not limited thereto, and the present invention is capable of various modifications and applications within the scope of equivalents interpreted and interpreted from the following claims. .

Unless otherwise indicated, nucleic acids are written in a 5'→3' orientation from left to right. Numerical ranges recited within the specification are inclusive of the numbers defining the range, including each integer or any non-integer fraction within the defined range.

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. Although any methods and materials similar or equivalent to those described herein can be used in the practice to test the present invention, the preferred materials and methods are described herein.

In one aspect, the present invention provides a method comprising: 1) a nucleic acid sequence A1, a nucleic acid sequence A2, a first toehold sequence A3, a nucleic acid sequence A4, and a second toehold sequence A5, wherein the nucleic acid sequence A4 and a second toehold sequence A5 is a complementary sequence capable of binding to all or part of a sequence of a target nucleic acid as a whole, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure, a first nucleic acid S1 (Substrat 1); 2) a second nucleic acid O (Output) sequentially comprising a sequence complementary to the nucleic acid sequence A4 and a sequence complementary to the first toehold sequence A3; 3) a third nucleic acid F1 (Fuel 1) sequentially comprising a sequence complementary to a part of the nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or part of the nucleic acid sequence A2; 4) sequentially comprising a nucleic acid sequence B1, a nucleic acid sequence B2, a third toehold sequence B3, a nucleic acid sequence B4 and a fourth toehold sequence B5, wherein the nucleic acid sequence B4 and the fourth toehold sequence B5 as a whole are the second nucleic acid O a fourth nucleic acid S2 (Substrate 2), which is a complementary sequence capable of binding to all or part of a sequence, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure; 5) a fifth nucleic acid U (Universal code) sequentially comprising a sequence complementary to the nucleic acid sequence B4 and a sequence complementary to the third toehold sequence B3; and 6) a sixth nucleic acid F2 (Fuel 2) sequentially comprising a sequence complementary to a part of the nucleic acid sequence B4, a sequence complementary to the third toehold sequence B3, and a sequence complementary to all or part of the nucleic acid sequence B2; It relates to a nucleic acid detection system comprising:

In one embodiment, the system of the present invention changes the fourth toehold sequence B5 of S1, F1, O and S2 according to the sequence of T (target nucleic acid), such as the sequence complementarity relationship of each nucleic acid sequence shown in FIG. 11 The sequences of , U and F2 can be fixed and used without modification as constructed by a technician, so that the sequence of the target nucleic acid can be unified with the U sequence, without changing the sequence of the nucleic acid detection construct of a complex structure (eg, fluorescent nanonucleic acid structure) It has the advantage of being able to detect various target nucleic acids.

In one aspect, the present invention provides a method comprising: 1) a nucleic acid sequence A1, a nucleic acid sequence A2, a first toehold sequence A3, a nucleic acid sequence A4, and a second toehold sequence A5, wherein the nucleic acid sequence A4 and a second toehold sequence A5 is a complementary sequence capable of binding to all or part of a sequence of a target nucleic acid as a whole, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure; a first nucleic acid S1; 2) a second nucleic acid O sequentially comprising a sequence complementary to the nucleic acid sequence A4 and a sequence complementary to the first toehold sequence A3; 3) a third nucleic acid F1 sequentially comprising a sequence complementary to a portion of the nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or a portion of the nucleic acid sequence A2; 4) sequentially comprising a nucleic acid sequence B1, a nucleic acid sequence B2, a third toehold sequence B3, a nucleic acid sequence B4 and a fourth toehold sequence B5, wherein the nucleic acid sequence B4 and the fourth toehold sequence B5 as a whole are the second nucleic acid O a fourth nucleic acid S2 that is a complementary sequence capable of binding to all or part of a sequence, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure; 5) a fifth nucleic acid U sequentially comprising a sequence complementary to nucleic acid sequence B4 and a sequence complementary to a third toehold sequence B3; and 6) a sixth nucleic acid F2 comprising sequentially a sequence complementary to a portion of nucleic acid sequence B4, a sequence complementary to a third toehold sequence B3 and a sequence complementary to all or a portion of nucleic acid sequence B2. It relates to a detection signal amplification system.

In one embodiment, the nucleic acid detection system of the present invention can amplify and detect a detection signal of a target nucleic acid.

In the present invention, the target nucleic acid (T) is complementary to S1O through a toehold mediated strand displacement reaction (S1T), and then with S1 through a toehold mediated strand displacement reaction with the third nucleic acid F1. Since the target sequence is released and reused by F1 complementary binding, its signal is first amplified, and the second nucleic acid O released by binding of the target nucleic acid with S1 complementarily binds with S2U through a toehold-mediated strand displacement reaction. After (S2O), the second nucleic acid O is released and reused by complementary binding of S2 and F2 through a toehold-mediated strand displacement reaction with the sixth nucleic acid F2, so that the signal thereof is amplified secondarily.

In one aspect, the present invention provides a method comprising: 1) a nucleic acid sequence A1, a nucleic acid sequence A2, a first toehold sequence A3, a nucleic acid sequence A4, and a second toehold sequence A5, wherein the nucleic acid sequence A4 and a second toehold sequence A5 is a complementary sequence capable of binding to all or part of a sequence of a target nucleic acid as a whole, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure; a first nucleic acid S1; 2) a second nucleic acid O sequentially comprising a sequence complementary to the nucleic acid sequence A4 and a sequence complementary to the first toehold sequence A3; 3) a third nucleic acid F1 sequentially comprising a sequence complementary to a portion of the nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or a portion of the nucleic acid sequence A2; 4) sequentially comprising a nucleic acid sequence B1, a nucleic acid sequence B2, a third toehold sequence B3, a nucleic acid sequence B4 and a fourth toehold sequence B5, wherein the nucleic acid sequence B4 and the fourth toehold sequence B5 as a whole are the second nucleic acid O a fourth nucleic acid S2 that is a complementary sequence capable of binding to all or part of a sequence, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure; 5) a fifth nucleic acid U sequentially comprising a sequence complementary to nucleic acid sequence B4 and a sequence complementary to a third toehold sequence B3; and 6) a sixth nucleic acid F2 sequentially comprising a sequence complementary to a portion of nucleic acid sequence B4, a sequence complementary to a third toehold sequence B3 and a sequence complementary to all or a portion of nucleic acid sequence B2. It is about a unified system.

In one embodiment, the target sequence may be converted to a second nucleic acid O in a first cycle, and to a fifth nucleic acid U (ie, a universal code having a given sequence) in a second cycle (see FIG. 1 ).

The present invention converts various different nucleotide sequences depending on the target into a universal code (U) having a single sequence completely independent of the target sequence (T), and uses the detection system for the universal code to various nucleotide sequences It has the advantage of being able to detect

In one aspect, the present invention provides a method comprising: 1) a nucleic acid sequence A1, a nucleic acid sequence A2, a first toehold sequence A3, a nucleic acid sequence A4, and a second toehold sequence A5, wherein the nucleic acid sequence A4 and a second toehold sequence A5 is a complementary sequence capable of binding to all or part of a sequence of a target nucleic acid as a whole, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure; a first nucleic acid S1; 2) a second nucleic acid O sequentially comprising a sequence complementary to the nucleic acid sequence A4 and a sequence complementary to the first toehold sequence A3; 3) a third nucleic acid F1 sequentially comprising a sequence complementary to a portion of the nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or a portion of the nucleic acid sequence A2; 4) sequentially comprising a nucleic acid sequence B1, a nucleic acid sequence B2, a third toehold sequence B3, a nucleic acid sequence B4 and a fourth toehold sequence B5, wherein the nucleic acid sequence B4 and the fourth toehold sequence B5 as a whole are the second nucleic acid O a fourth nucleic acid S2 that is a complementary sequence capable of binding to all or part of a sequence, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure; 5) a fifth nucleic acid U sequentially comprising a sequence complementary to nucleic acid sequence B4 and a sequence complementary to a third toehold sequence B3; and 6) a sixth nucleic acid F2 comprising sequentially a sequence complementary to a portion of nucleic acid sequence B4, a sequence complementary to a third toehold sequence B3 and a sequence complementary to all or a portion of nucleic acid sequence B2. It relates to a composition for detection.

In one embodiment, the target nucleic acid may be a biomarker such as a short miRNA in vivo, a genetic biomarker in a liquid biopsy, or a small non-coding RNA (sncRNA) such as a miRNA.

In one embodiment, the second nucleic acid O and the first nucleic acid S1 complementarily bind to form a double strand (S10), and complementarily bind with the fifth nucleic acid U and the fourth nucleic acid S2 to form a double strand (S2U) may be forming.

In one embodiment, the second toehold sequence A5 in S10 and the fourth toehold sequence B5 in S2U may be exposed as a single strand.

In one embodiment, the second toehold sequence A5 or the fourth toehold sequence B5 may comprise 4 to 8 nucleotides.

In one embodiment, the third nucleic acid F1 or the sixth nucleic acid F2 may have a hairpin structure.

In one embodiment, the stem portion of the third nucleic acid F1 or the sixth nucleic acid F2 (the double bond portion of the hairpin structure may include 6 to 12 nucleotides).

In one embodiment, the first toehold sequence A3 or the third toehold sequence B3 may comprise 4 to 12 nucleotides.

In one embodiment, the first nucleic acid S1 may further comprise a quencher.

In one embodiment, the quencher is Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa black FQ, Iowa black RQ , may be one or more selected from the group IRDye QC-1.

In one embodiment, the second nucleic acid O and the fifth nucleic acid U may include a fluorescent material.

In one embodiment, the fluorescent material is fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, Cyanine-based dyes and thiadicarbocyanine (thiadicarbocyanine) may be at least one selected from the group consisting of.

In one embodiment, it is possible to amplify the signal of the presence of a target nucleic acid as a fifth nucleic acid U released through a toehold mediated strand displacement reaction characterized by nulls and self-assembly.

In one embodiment, a composition for detecting the fifth nucleic acid U may be further included.

In one embodiment, it may further include a fluorescent nucleic acid nanostructure comprising a single-stranded probe comprising a nucleic acid complementary to the fifth nucleic acid U and a fluorescent substance, and graphene oxide.

In one embodiment, the composition for detecting the fifth nucleic acid U is a nucleic acid sequence complementary to the fifth nucleic acid U, a primer pair that specifically recognizes the nucleic acid sequence and a fragment of the complementary sequence, or a probe, or a primer pair, and It may include a probe.

In one embodiment, the detection of the fifth nucleic acid U is polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive polymerase chain reaction (Competitive RT-PCR), nuclease protection Analysis (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, can be detected by an analysis method including a DNA chip, but a single strand comprising a nucleic acid complementary to the fifth nucleic acid U and a fluorescent material It is more preferable to detect with a fluorescent nucleic acid nanostructure including a probe, and a fluorescent nucleic acid nanostructure for nucleic acid detection including graphene oxide-graphene oxide complex.

In one embodiment, the fluorescent nucleic acid nanostructure for nucleic acid detection-graphene oxide complex can detect fluorescence by a fluorescence resonance energy transfer phenomenon.

As used herein, the term "fluorescent nanonucleic acid construct" includes a single-stranded probe comprising a nucleic acid and a fluorescent substance complementary to a target nucleic acid, a construct composed of the probes, or the probes bound to a target nucleic acid.

As used herein, the term "fluorescent nucleic acid nanostructure/graphene oxide complex" or "graphene fluorescent nucleic acid nanostructure" refers to a structure or complex in which the fluorescent nucleic acid nanostructure is attached on graphene oxide.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding to mRNA, and is labeled to check the presence or absence of a specific nucleic acid. can The probe may be manufactured in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like, but in the present invention, it refers to a single-stranded nucleic acid.

In one embodiment, the fluorescent nucleic acid nanostructure for nucleic acid detection-graphene oxide complex comprises a first single-stranded probe comprising a nucleic acid and a fluorescent substance complementary to the universal code A converted and released for the target a; a second single-stranded probe comprising a nucleic acid complementary to the universal code B and a fluorescent substance converted and released for the target b; a third single-stranded probe comprising a nucleic acid and a fluorescent substance complementary to the universal code C converted and released for the target c; and a fourth single-stranded probe comprising a sequence complementary to the first single-stranded probe, a sequence complementary to the second single-stranded probe, and a sequence complementary to the third single-stranded probe.

In one embodiment, the fluorescent materials included in the single-stranded probes may be fluorescent materials of different wavelength bands.

In one embodiment, the fluorescent nucleic acid nanostructure for nucleic acid detection-graphene oxide complex fluorescent nucleic acid nanostructure has any one of a first single-stranded probe, a second single-stranded probe, and a third single-stranded probe attached to graphene oxide, It may have a tetrahedral structure on graphene oxide.

In one embodiment, the fluorescent nucleic acid nanostructure for nucleic acid detection-graphene oxide complex fluorescent nucleic acid nanostructure has a first single-stranded probe, a second single-stranded probe and a third single-stranded probe of the fluorescent nucleic acid nanostructure attached to graphene oxide The fluorescent nucleic acid nanostructure may have a triangular columnar structure on graphene oxide.

In one embodiment, the fluorescent nucleic acid nanostructure for nucleic acid detection-graphene oxide complex fluorescent nucleic acid nanostructure has a first single-stranded probe, a second single-stranded probe and a third single-stranded probe attached to graphene oxide, graphene oxide It may have a triangular prism structure.

In one embodiment, graphene oxide (GO) is a fluorescence-limiting chemical (Universal Quencher: UQ) that can selectively block fluorescence by fluorescence resonance energy transfer, and the target nucleic acid and the single-stranded probe of the present invention When a double bond is formed by bonding, the probe (or fluorescent nanonucleic acid structure) moves away from the graphene oxide, so that the emission of the fluorescent material can be detected. That is, graphene oxide is more easily attached to a single-stranded base sequence than a double-stranded base sequence, and according to the fluorescence resonance energy transfer, when the distance between the graphene oxide and the fluorescent material of the fluorescent nanonucleic acid structure is close, the fluorescence is blocked. , using the property that fluorescence can be detected again when the distance increases, multiple target nucleotide sequences in probes containing a fluorescent material combine with a sequence complementary to each of the probes to form a double bond from graphene oxide By emitting fluorescence as the distance increases, multiple targets can be recognized and quantified at once.

In one embodiment, when the target nucleic acid is complementary to the second threshold sequence A5 of the first nucleic acid S1, the second nucleic acid O is released through a branch migration reaction and the first threshold sequence A3 is exposed become; the first toehold sequence A3 of the first nucleic acid S1 bound to the target nucleic acid binds to the third nucleic acid F1 to release the target nucleic acid through a branch point shift reaction; the released second nucleic acid O complementarily binds to the fourth threshold sequence B5 of the fourth nucleic acid S2 to release the fifth nucleic acid U through a branch point shift reaction; The third toehold sequence B3 of the fourth nucleic acid S2 bound to the second nucleic acid O may be bound to the sixth nucleic acid F2 to release the second nucleic acid O through a branch point shift reaction.

In one embodiment, the released target nucleic acid is re-reacted with the second toehold sequence A5 of the second nucleic acid O and the first nucleic acid S1 (S10), which are complementary to form a double strand, and are released from the above The second nucleic acid O may be re-reacted with the fourth toehold sequence B5 of the fourth nucleic acid S2 and the fifth nucleic acid U (S2U) that are complementary to each other to form a double strand.

In one aspect, the present invention provides a method comprising: 1) a nucleic acid sequence A1, a nucleic acid sequence A2, a first toehold sequence A3, a nucleic acid sequence A4, and a second toehold sequence A5, wherein the nucleic acid sequence A4 and a second toehold sequence A5 is a complementary sequence capable of binding to all or part of a sequence of a target nucleic acid as a whole, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure; a first nucleic acid S1; 2) a second nucleic acid O sequentially comprising a sequence complementary to the nucleic acid sequence A4 and a sequence complementary to the first toehold sequence A3; 3) a third nucleic acid F1 sequentially comprising a sequence complementary to a portion of the nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or a portion of the nucleic acid sequence A2; 4) sequentially comprising a nucleic acid sequence B1, a nucleic acid sequence B2, a third toehold sequence B3, a nucleic acid sequence B4 and a fourth toehold sequence B5, wherein the nucleic acid sequence B4 and the fourth toehold sequence B5 as a whole are the second nucleic acid O a fourth nucleic acid S2 that is a complementary sequence capable of binding to all or part of a sequence, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure; 5) a fifth nucleic acid U sequentially comprising a sequence complementary to nucleic acid sequence B4 and a sequence complementary to a third toehold sequence B3; and 6) a sixth nucleic acid F2 sequentially comprising a sequence complementary to a portion of nucleic acid sequence B4, a sequence complementary to a third toehold sequence B3 and a sequence complementary to all or a portion of nucleic acid sequence B2. It relates to a kit for detecting nucleic acids.

In one aspect, the present invention provides a method comprising: 1) a nucleic acid sequence A1, a nucleic acid sequence A2, a first toehold sequence A3, a nucleic acid sequence A4, and a second toehold sequence A5, wherein the nucleic acid sequence A4 and a second toehold sequence A5 is a complementary sequence capable of binding to all or part of a sequence of a target nucleic acid as a whole, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure; a first nucleic acid S1; 2) a second nucleic acid O sequentially comprising a sequence complementary to the nucleic acid sequence A4 and a sequence complementary to the first toehold sequence A3; 3) a third nucleic acid F1 sequentially comprising a sequence complementary to a portion of the nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or a portion of the nucleic acid sequence A2; 4) sequentially comprising a nucleic acid sequence B1, a nucleic acid sequence B2, a third toehold sequence B3, a nucleic acid sequence B4 and a fourth toehold sequence B5, wherein the nucleic acid sequence B4 and the fourth toehold sequence B5 as a whole are the second nucleic acid O a fourth nucleic acid S2 that is a complementary sequence capable of binding to all or part of a sequence, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure; 5) a fifth nucleic acid U sequentially comprising a sequence complementary to nucleic acid sequence B4 and a sequence complementary to a third toehold sequence B3; and 6) a sixth nucleic acid F2 comprising sequentially a sequence complementary to a portion of nucleic acid sequence B4, a sequence complementary to a third toehold sequence B3 and a sequence complementary to all or a portion of nucleic acid sequence B2. It relates to a composition or kit for amplifying a detection signal.

In one aspect, the present invention provides a method comprising: 1) a nucleic acid sequence A1, a nucleic acid sequence A2, a first toehold sequence A3, a nucleic acid sequence A4, and a second toehold sequence A5, wherein the nucleic acid sequence A4 and a second toehold sequence A5 is a complementary sequence capable of binding to all or part of a sequence of a target nucleic acid as a whole, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure; a first nucleic acid S1; 2) a second nucleic acid O sequentially comprising a sequence complementary to the nucleic acid sequence A4 and a sequence complementary to the first toehold sequence A3; 3) a third nucleic acid F1 sequentially comprising a sequence complementary to a portion of the nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or a portion of the nucleic acid sequence A2; 4) sequentially comprising a nucleic acid sequence B1, a nucleic acid sequence B2, a third toehold sequence B3, a nucleic acid sequence B4 and a fourth toehold sequence B5, wherein the nucleic acid sequence B4 and the fourth toehold sequence B5 as a whole are the second nucleic acid O a fourth nucleic acid S2 that is a complementary sequence capable of binding to all or part of a sequence, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure; 5) a fifth nucleic acid U sequentially comprising a sequence complementary to nucleic acid sequence B4 and a sequence complementary to a third toehold sequence B3; and 6) a sixth nucleic acid F2 sequentially comprising a sequence complementary to a portion of nucleic acid sequence B4, a sequence complementary to a third toehold sequence B3, and a sequence complementary to all or a portion of nucleic acid sequence B2, It relates to a composition for universal code conversion, which converts a target nucleic acid into a fifth nucleic acid U, which is a predetermined universal code, through a hold-mediated strand displacement reaction.

In one embodiment, the fourth threshold sequence B5 of the first nucleic acid S1, the second nucleic acid O, the third nucleic acid F1 and the fourth nucleic acid S2 may be changed according to the target nucleic acid, and the sixth nucleic acid F2 and the fifth nucleic acid U may be fixed to a predetermined sequence irrespective of the target nucleic acid.

In one aspect, the present invention provides a method comprising: 1) a nucleic acid sequence A1, a nucleic acid sequence A2, a first toehold sequence A3, a nucleic acid sequence A4, and a second toehold sequence A5, wherein the nucleic acid sequence A4 and a second toehold sequence A5 is a complementary sequence capable of binding to all or part of a sequence of a target nucleic acid as a whole, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure; a first nucleic acid S1; 2) a second nucleic acid O sequentially comprising a sequence complementary to the nucleic acid sequence A4 and a sequence complementary to the first toehold sequence A3; 3) a third nucleic acid F1 sequentially comprising a sequence complementary to a portion of the nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or a portion of the nucleic acid sequence A2; 4) sequentially comprising a nucleic acid sequence B1, a nucleic acid sequence B2, a third toehold sequence B3, a nucleic acid sequence B4 and a fourth toehold sequence B5, wherein the nucleic acid sequence B4 and the fourth toehold sequence B5 as a whole are the second nucleic acid O a fourth nucleic acid S2 that is a complementary sequence capable of binding to all or part of a sequence, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure; 5) a fifth nucleic acid U sequentially comprising a sequence complementary to nucleic acid sequence B4 and a sequence complementary to a third toehold sequence B3; and 6) a sixth nucleic acid F2 sequentially comprising a sequence complementary to a portion of nucleic acid sequence B4, a sequence complementary to a third toehold sequence B3, and a sequence complementary to all or a portion of nucleic acid sequence B2, To a universal code conversion kit, which converts a target nucleic acid into a fifth nucleic acid U, which is a predetermined universal code, through a hold-mediated strand displacement reaction.

In one aspect, the present invention provides 1) a nucleic acid sequence A4 and a second threshold sequence A5 of the first nucleic acid S1 in the nucleic acid detection system of the present invention are changed to a complementary sequence capable of binding to all or part of a target nucleic acid sequence, thereby altering the fourth toehold sequence B5 of the second nucleic acid O, the third nucleic acid F1 and the fourth nucleic acid S2; and 2) detecting a fifth nucleic acid U released via a toehold mediated strand displacement reaction.

In one embodiment, the sixth nucleic acid F2 and the fifth nucleic acid U, which is the universal code, may be a sequence-fixed predetermined nucleic acid.

In one embodiment, the released fifth nucleic acid U can be detected using a fluorescent nucleic acid nanostructure specific for the released fifth nucleic acid U and graphene oxide.

In one aspect, the present invention relates to a sequence unification method using the universal code conversion system of the present invention.

In one aspect, the present invention provides a hybrid product of a third nucleic acid F1 and a sixth nucleic acid F2; a double strand (S10) to which a second nucleic acid O and a first nucleic acid S1 are complementarily bound; And it relates to a biosensor for detecting a target nucleic acid comprising a double-stranded (S2U) complementary to the fifth nucleic acid U and the fourth nucleic acid S2.

In one embodiment, it may further include a fluorescent nucleic acid nanostructure comprising a single-stranded probe comprising a nucleic acid complementary to the fifth nucleic acid U and a fluorescent substance, and graphene oxide.

In one aspect, the present invention relates to a system for amplifying a nucleic acid detection or detection signal for each of the miRNAs of Table 1.

In one embodiment, the first nucleic acid S1, the second nucleic acid O, the third nucleic acid F1, the fourth nucleic acid S2, the fifth nucleic acid U and the sixth nucleic acid F2 for each of the miRNAs may be as shown in Table 3 .

In one embodiment, the nucleic acid detection or detection signal amplification system of the sequence shown in Table 3 may be cancer specific.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1. Universal Code Conversion System Fabrication and Optimization

1-1. Universal Code Conversion System Configuration

To amplify and detect the target sequence by targeting miR-21 and converting it into a universal code having a specific sequence, a scaffold mediated strand displacement reaction (Yurke, Bernard (2000)) Based on "A DNA-fuelled molecular machine made of DNA". Nature. 406 (6796): 605-8.), a universal code conversion system consisting of two cycles was fabricated. Specifically, the universal code conversion system proceeds by a two-cycle system, where the target miRNA is first converted to the output sequence attached to Substrate 1 (S1) through the first cycle system, and then moves to the second cycle system and moves to Substrate 2 It is converted into the form of the universal code (U) attached to (S2), and in each cycle, the target is a system that can be reused by the fuel sequence. That is, in Cycle 1, the target sequence, miR-21, is invading the toehold part of the complementary bound S1 (Substrate 1) and the output sequence (original strand) (the 3' part of S1 to which the output sequence is not bound, miRNA toehold). input as a strand and the output sequence goes to cycle 2; The target sequence bound to S1 is separated from S1 as the Fuel 1 sequence (F1) is input as an invading strand to the Fuel 1 toehold part (the part where the 3' of the target sequence miRNA and the S1 sequence is not bound, Fuel toehold); And again, it is input as an invading strand to the structure in which S1 and the output sequence are combined and recycled (Cycle 1 in FIG. 1). In addition, in cycle 2, the output sequence transferred from cycle 1 is input as an invading strand to the toehold part of the universal code complementary to the S2 sequence (Substrate 2) (the 3' part of the S2 sequence that is not bound to the universal code). code comes off S2; And the Fuel 2 sequence (F2) is input as an invading strand to the Fuel 2 toehold portion of the Output sequence coupled with S2 (the portion where the 3' and S2 sequences of the Output sequence are not coupled), so that the Output sequence is separated from S2 and re-combined with S2 In the complementary combined universal code, it is input again to the toehold part and recycled (Cycle 2 in FIG. 1). For this, S1 and S2, Output (O), Fuel 1 and 2 (F1 and F2), and universal code (U) for miR-21 of the sequence shown in Table 1 through the Nupack program It was designed in consideration of the thermodynamic reaction relationship (FIG. 2), and sequences under various conditions were designed for sequence optimization experiments (Table 2). The designed and manufactured sequences were stored in TE buffer, and S1, Output, S2, and universal codes were double-stranded in TE buffer with a salt concentration of 200 mM using Bio Rad MyCycler Thermal Cycler PCR (Bio Rad, Hercules, California, USA). was combined with

miRNA 5'-Sequence-3' SEQ ID No. miR-200b-3p UAA UAC UGC CUG GUA AUG AUG A One miR-200c UAA UAC UGC CGG GUA AUG AUG GA 2 miR-let 7a UGA GGU AGU AGG UUG UAU AGU U 3 miR-let 7 c UGA GGU AGU AGG UUG UAU GGU U 4 miR-let 7d AGA GGU AGU AGG UUG CAU AGU U 5 miR-let 7 f UGA GGU AGU AGA UUG UAU AGU U 6 miR-191 CAA CGG AAU CCC AAA AGC AGC UG 7 miR-26a UUC AAG UAA UCC AGG AUA GGC U 8 miR-342-3p UCU CAC ACA GAA AUC GCA CCC GU 9 miR-1826 AUU GAU CAU CGA CAC UUC GAA CGC AAU 10 miR-1979 CUC CCA CUG CUU CAC UUG ACU A 11 miR-21 UAG CUU AUC AGA CUG AUG UUG A 12

*In the case of miR-21, the initial tendency of the system of the present invention was confirmed by attaching a Cy3 dye to the 3'-end.

Name of strand version 5'-Sequence-3' SEQ ID
No. O
(output) m 8 toehold CAG ACT GAT GTT GAA GTA CCC CAG TGT T 13 m 10 toehold GAC TGA TGT TGA AGT ACC CCA GTG TT 14 m 12 toehold CTG ATG TTG AAG TAC CCC AGT GTT 15 F1
(Fuel 1) No loop 8 stem GAC TGA TGT TGA AGT ACC GAC GTA CG 16 loop 8 stem GAC TGA TGT TGA AGT ACC GAC GTA CGT ATA CGT ACG 17 No loop 10 stem GAC TGA TGT TGA AGT ACC CAG ACG TAC G 18 loop 10 stem GAC TGA TGT TGA AGT ACC CAG ACG TAC GTA TAC GTA CG 19 S1
(substrate 1) 8 stem GAC GTA CGT ATA CGT ACG TCG GTA CTT CAA CAT CAG TCT GAT AAG CTA 20 10 stem CAG ACG TAC GTA TAC GTA CGT CTG GGT ACT TCA ACA TCA GTC TGA TAA GCT A 21 O
(output) F 4 toehold GAC TGA TGT TGA AGT A CCA GTG TT 22 F 6 toehold GAC TGA TGT TGA AGT ACC CCA GTG TT 23 F 8 toehold GAC TGA TGT TGA AGT ACC GA CCA GTG TT 24 F1
(Fuel 1) F 4 toehold GAC TGA TGT TGA AGT A CAG ACG TAC GTA TAC GTA CG 25 F 6 toehold GAC TGA TGT TGA AGT ACC CAG ACG TAC GTA TAC GTA CG 26 F 8 toehold GAC TGA TGT TGA AGT ACC GA CAG ACG TAC GTA TAC GTA CG 27 S1
(substrate 1) F 4 toehold CAG ACG TAC GTA TAC GTA CGT CTG T ACT TCA ACA TCA GTC TGA TAA GCT A 28 F 6 toehold CAG ACG TAC GTA TAC GTA CGT CTG GGT ACT TCA ACA TCA GTC TGA TAA GCT A 29 F 8 toehold CAG ACG TAC GTA TAC GTA CGT CTG TC GGT ACT TCA ACA TCA GTC TGA TAA GCT A 30

Example 2. Universal Code Sequence Optimization

For the optimization of the universal code sequence, the toehold sequence of the Output in S1 and S2, Output, Fuel 1 and 2, and universal codes (U) (Table 2) for miR-21 prepared above (Table 2) The single-stranded portion of S1 that is not bound, the portion into which the miRNA enters, shown at the bottom of Figure 3a) the number (4, 6 or 8), the presence or absence of a ring of the Fuel sequence, the number of stems, and the Fuel toehold sequence of S1 (target A single sequence was revealed because the sequence and S1 were not combined, so the difference in the reaction according to the number of parts where Fuel 1 entered (shown at the bottom of FIG. 3c) was confirmed by electrophoresis. For electrophoresis, the sample was loaded on 15% poly acryl amide gel, and Bio Rad PowerPac basic (Bio Rad, Hercules, CA, USA) and Bio Rad CRITERION Cell (Bio Rad, Hercules, California, USA) were used. It was carried out under TBE buffer conditions for 1 hour at 110 V using the electrophoresis, and the temperature was maintained at 4 °C during electrophoresis. Each sample was reacted at 200 nM based on the molar concentration, and the reaction time and temperature were set to 6 hours and 25°C. During electrophoresis, each sample was loaded in a volume of 10 μ, and 30 to 50 ng of each sample was loaded by weight. After electrophoresis, the polyacrylamide gel was stained with Gel Red for 30 minutes and images were obtained using FluoroBox (NEO Science, Seoul, Korea). For quantitative analysis of the reaction progress, electrophoresis was performed using the Imagej tool. The band strength was analyzed. As a result of optimizing the reaction temperature or the ratio between the components of the time universal code system in the experimental condition, the reaction progress of cycle 1 was confirmed under the reaction temperature condition. In the case of rows 2 and 3, it seemed that the reaction progress could increase as the temperature increased, but there was no significant difference, so the experiment was conducted at 25° C. (FIG. 3a). In addition, as for the reaction time, it was confirmed that almost all of them were converted to the S1F1 band in column 3 after the time, and the reaction time was also measured after the reaction progressed for 6 hours ( FIG. 3b ). In addition, the reaction progress through the electrophoresis image was analyzed using Equation 1 below for the intensity of the band obtained using the image J program.

A = (intensity of the resulting band of the sample in which the reaction was performed) / (band intensity of the resultant band of the control group); and

B = (band intensity before the reaction of the sample in which the reaction was performed) / (band intensity of the control band before the reaction).

As a result, when the number of toehold sequences was 4, S10-S1m reaction hardly occurred, but S10-S1F1 was about 19% and S1m-S1F1 was about 47% in 6, and S10-S1F1 was 42 in 8. % and S1m-S1F1 were found to be 58% (Fig. 4a). In addition, when there was no ring in the fuel sequence, the S1O-S1F1 reaction proceeded very well, and when the number of stems was 8 (S1O-S1F1 14% and S1m-S1F1 22%), when there were 10 (S1O-S1F1 13%) and S1m-S1F1 37%) showed that the reaction proceeded more stably ( FIG. 4b ). In addition, the reaction progress of S1O-S1F1 and S1m-S1F1 was 9% and 37%, respectively, when the number of fuel toeholds of S1 was 4, 10% and 41% when 6, and 14% and 42% when 8 ( Fig. 4c). Accordingly, the fuel sequence was selected as 10 stem sequences and 6 toehold sequences in the ring structure.

Example 3. Confirmation of Universal Code Conversion

In the universal code conversion process of the present invention, the reaction progress by concentration in each step of Cycle 1 and Cycle 2 was confirmed by the electrophoresis method, the detection limit was calculated by the following Equation 2, and the detection signal amplification rate was calculated as the following Equation 3 Calculated.

s = standard deviation of the response; and

S = slope of the calibration curve (linear).

As a result, the progress of the reaction with respect to the concentration was shown to be in a direct proportional relationship with each step, and although there was a difference according to the reaction rate, it was confirmed that the reaction progress as a whole over time had an S-shaped figure (FIG. 5). Specifically, columns 1 and 2 of the electrophoretic image of FIG. 5 show the transition to the next stage target, columns 3 and 4 show the difference in response depending on the presence or absence of the target, and the band intensity is measured with the Image J tool, and the above equation When calculated as 1, it was found that about 73% of the reaction proceeded in column 1 of FIG. 5a, and about 94% progressed in column 2 of FIG. 5b, and about 93% in column 1 of FIG. appeared to have progressed. In addition, a difference in reaction progress by the target in columns 3 and 4 of FIG. 5A was 82%, and in columns 3 and 4 of FIG. 5B, a difference in reaction progress of 64% was confirmed. In addition, as a result of confirming the reaction progress of the universal code according to the miRNA concentration, it was confirmed that the average amplification was about 3.95 times in Cycle 1 for 6 hours and about 1.24 times in Cycle 2, and in the entire cycle, it was amplified by an average of 2.13 times based on 6 hours. , it was found that the amplification rate increased by 4.13 times when the reaction was carried out for up to 12 hours (FIG. 6). Through this, it seems that the overall reaction time increased as both cycles were rotated, and the detection limit of the system was calculated to be 18.5 nM.

Example 4. Application to various target RNAs

In order to confirm the specificity of the universal code system, S1 and S2, Output, Fuel 1 and 2, and universal codes (U) for the miRNAs in Table 1 were prepared as in the Examples (Table 1). 3). Thereafter, reaction progress when a total of 12 types of miRNAs were injected against the universal code of miR-21 was analyzed by fluorescence. Specifically, the fluorescence sample was reacted in TE buffer with 200 mM salt concentration, and FAM (Fluorescein phosphoramidite, excitation wavelength) was used for 12 hours using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, California, USA) to measure fluorescence kinetics. 485 nm, emission wavelength 525 nm), Cy 3 (Cyanine-3, excitation wavelength 540 nm, emission wavelength 570 nm), and Cy 5 (Cyanine-5, excitation wavelength 640 nm, emission wavelength 670 nm) conditions were confirmed. Each sample was tested at a concentration of 100 nM and a volume of 100 μ, and all experiments were performed three times by the same experimenter under the same conditions. The reaction of each cycle system is to inject the target miRNAs miR-21 and F1 into the S10 sequence (the state in which the S1 sequence and the O sequence are combined), respectively, or the O and F2 sequences in the S2U sequence (the state where the S1 sequence and the U sequence are combined), respectively. was made in the form of injection. In the case of the whole cycle system, it was done by injecting F1, F2, and miRNA by concentration into a sample containing S10 and S2U at the same time. Finally, in the confirmation of universal code conversion for 12 species, in the presence of S1O, S2U, F1 and F2, when miRNA was inserted and when not, and when S2 with a quencher was put back in, the amount of light blocked through the amount of universal code The conversion was confirmed. In addition, the reaction progress of the Cycle 1 part of the universal code conversion system for each of the 12 miRNAs and the reaction progress of the Cycle 2 part were confirmed as in Examples 2 and 3 by electrophoresis.

miRNA Strand name 5'-Sequence-3' SEQ ID
No. miR-
200b-3p S1
substrate 1) CAG ACG TAC G TAT A CGT ACG TCT G GGT ACT TCA TCA TTA CCA GGC AGT ATT A 31 O
(output) TGG TAA TGA TGA AGT ACC CCA GTG TT 32 F1
(fuel 1) TGG TAA TGA TGA AGT ACC CAG ACG TAC G TAT A CGT ACG 33 S2
(substrate 2) ( Quencher/normal ) - GGT CTA GGA A TAT A TTC CTA GAC C ACA CCA AAC ACT GGG GTA CTT CAT CAT T 34 miR-
200c S1
(substrate 1) CAG ACG TAC G TAT A CGT ACG TCT G GGT ACT TCC ATC ATT ACC CGG CAG TAT T 35 O
(output) GGT AAT GAT GGA AGT ACC CCA GTG TT 36 F1
(fuel 1) GGT AAT GAT GGA AGT ACC CAG ACG TAC GTA TAC GTA CG 37 S2
(substrate 2) ( Quencher/normal ) - GGT CTA GGA A TAT A TTC CTA GAC C ACA CCA AAC ACT GGG GTA CT TCCATCAT 38 miR-
let 7a S1
(substrate 1) CAG ACG TAC G TAT A CGT ACG TCT G GGT ACT AAC TAT ACA ACC TAC TAC CTC A 39 O
(output) GGT TGT ATA GTT AGT ACC CCA GTG TT 40 F1
(fuel 1) GGT TGT ATA GTT AGT ACC CAG ACG TAC G TAT A CGT ACG 41 S2
(substrate 2) ( Quencher/normal ) - GGT CTA GGA A TAT A TTC CTA GAC C ACA CCA AAC ACT GGG GTA CTA ACT ATA C 42 miR-
let 7c S1
(substrate 1) CAG ACG TAC G TAT A CGT ACG TCT G GGT ACT AAC CAT ACA ACC TAC TAC CTC A 43 O
(output) GGT TGT ATG GTT AGT ACC CCA GTG TT 44 F1
(fuel 1) GGT TGT ATG GTT AGT ACC CAG ACG TAC G TAT A CGT ACG 45 S2
(substrate 2) ( Quencher/normal ) - GGT CTA GGA A TAT A TTC CTA GAC C ACA CCA AAC ACT GGG GTA CTA ACC ATA C 46 miR-
let 7d S1
(substrate 1) CAG ACG TAC G TAT A CGT ACG TCT G GGT ACT AAC TAT GCA ACC TAC TAC CTC T 47 O
(output) GGT TGC ATA GTT AGT ACC CCA GTG TT 48 F1
(fuel 1) GGT TGC ATA GTT AGT ACC CAG ACG TAC G TAT A CGT ACG 49 S2
(substrate 2) ( Quencher/normal ) - GGT CTA GGA A TAT A TTC CTA GAC C ACA CCA AAC ACT GGG GTA CTA ACC ATA C 50 miR-
let 7f S1
(substrate 1) CAG ACG TAC G TAT A CGT ACG TCT G GGT ACT AAC TAT ACA ATC TAC TAC CTC A 51 O
(output) GAU UGU AUA GUU AGT ACC CCA GTG TT 52 F1
(fuel 1) GAU UGU AUA GUU AGT ACC CAG ACG TAC G TAT A CGT ACG 53 S2
(substrate 2) ( Quencher/normal ) - GGT CTA GGA A TAT A TTC CTA GAC C ACA CCA AAC ACT GGG GTA CTA ACT ATA C 54 miR-
191 S1
(substrate 1) CAG ACG TAC G TAT A CGT ACG TCT G GGT ACT CAG CTG CTT TTG GGA TTC CGT T 55 O
(output) CAA AAG CAG CTG AGT ACC CCA GTG TT 56 F1
(fuel 1) CAA AAG CAG CTG AGT ACC CAG ACG TAC G TAT A CGT ACG 57 S2
(substrate 2) ( Quencher/normal ) - GGT CTA GGA A TAT A TTC CTA GAC C ACA CCA AAC ACT GGG GTA CTC AGC TGC T 58 miR-
26a S1
(substrate 1) CAG ACG TAC G TAT A CGT ACG TCT G GGT ACT AGC CTA TCC TGG ATT ACT TGA A 59 O
(output) CCA GGA TAG GCT AGT ACC CCA GTG TT 60 F1
(fuel 1) CCA GGA TAG GCT AGT ACC CAG ACG TAC G TAT A CGT ACG 61 S2
(substrate 2) ( Quencher/normal ) - GGT CTA GGA A TAT A TTC CTA GAC C ACA CCA AAC ACT GGG GTA CT AGC CTA TC 62 miR-
342-3p S1
(substrate 1) CAG ACG TAC G TAT A CGT ACG TCT G GGT ACT ACG GGT GCG ATT TCT GTG TGA G 63 O
(output) AAT CGC ACC CGT AGT ACC CCA GTG TT 64 F1
(fuel 1) AAT CGC ACC CGT AGT ACC CAG ACG TAC G TAT A CGT ACG 65 S2
(substrate 2) ( Quencher/normal ) - GGT CTA GGA A TAT A TTC CTA GAC C ACA CCA AAC ACT GGG GTA CT ACG GGT GC 66 miR-
1826 S1
(substrate 1) CAG ACG TAC G TAT A CGT ACG TCT G GGT ACT ATT GCG TTC GAA GTG TCG ATG A 67 O
(output) TTC GAA CGC AAT AGT ACC CCA GTG TT 68 F1
(fuel 1) TTC GAA CGC AAT AGT ACC CAG ACG TAC GTA TAC GTA CG 69 S2
(substrate 2) ( Quencher/normal ) - GGT CTA GGA A TAT A TTC CTA GAC C ACA CCA AAC ACT GGG GTA CT ATTGCGTT 70 miR-
1979 S1
(substrate 1) CAG ACG TAC G TAT A CGT ACG TCT G GGT ACT TAG TCA AGT GAA GCA GTG GGA G 71 O
(output) TTC ACT TGA CTA AGT ACC CCA GTG TT 72 F1
(fuel 1) TTC ACT TGA CTA AGT ACC CAG ACG TAC GTA TAC GTA CG 73 S2
(substrate 2) ( Quencher/normal ) - GGT CTA GGA A TAT A TTC CTA GAC C ACA CCA AAC ACT GGG GTA CT TAGTCA AG 74 miR-21 S1
(substrate 1) ( Quencher/normal ) - CAG ACG TAC G TAT A CGT ACG TCT G GGT ACT TCA ACA TCA GTC TG ATA AGC TA 75 O
(output) GAC TGA TGT TGA AGT ACC CCA GTG TT - ( FAM/normal ) 76 F1
(fuel 1) GAC TGA TGT TGA AGT ACC CAG ACG TAC G TAT A CGT ACG 77 S2
(substrate 2) ( Quencher/normal ) - GGT CTA GGA A TAT A TTC CTA GAC C ACA CCA AAC ACT GGG GTA CT TCA ACA TC 78 Universal U
(universal code) T ACC CCA GTG TT TGG TGT GTTT - ( Cy5/normal ) 79 F2 (fuel 2) T ACC CCA GTG TT TGG TGT GGT CTA GGA A TAT A TTC CTA G 80

As a result, compared to when other miRNAs were injected into the universal code of miR-21, when miR-21 was injected, there was an average of about 7-fold or more difference in reaction progress ( FIG. 7a ). This means that the universal code for a specific target has a detectability specific to the target. A T-test was performed to analyze the significance accordingly, and it was confirmed that there was significance with a p-value of 0.01 or less for all miRNAs.

In addition, as a result of confirming the conversion and detection ability of the universal code system to the universal code system for each of the 12 target miRNAs, the fluorescence data of the sample injected with miRNA and the sample without the injection according to each coding system were clearly confirmed. A discriminable result difference was shown, and both showed a significant difference in the significance test through the T test (FIG. 7b). In addition, the electrophoresis results of the universal code system for each target miRNA also showed that each of the 12 target miRNAs was well converted into a universal code having a single sequence ( FIGS. 8 and 9 ).

Example 5 Graphene Sensor for Nucleic Acid Detection of Universal Code

Next, the fluorescence value according to the concentration was confirmed using the graphene sensor for nucleic acid detection of the present invention using the universal code. Samples were prepared in a TE buffer with a salt concentration of 200 mM, so that the concentration of the nucleic acid detection system was 100 nM, respectively, and the target nucleic acid (EGFR del 19 mutant) was added, followed by incubation for 30 minutes. After the TNT system (graphene biosensor) was injected at the same concentration, graphene oxide was added. Thereafter, fluorescence values were measured by FAM (Fluorescein phosphoramide, excitation wavelength 485 nm, emission wavelength 525 nm) using a SpectraMax M5 microplate. Table 4 shows the target nucleic acid (EGFR del 19 mutant) used and the respective probe sequences of the TNT system.

As shown in FIG. 10 , it was confirmed that the fluorescence value increased to the sigmoidal type as the target concentration was changed to 5, 10, 20, 50, 100, 150 and 200 nM, which resulted in the successful conversion of the target shape and It means some amplification.

miRNA Strand name 5'-Sequence-3' SEQ ID
No. EGFR del 19 S1
(substrate 1) CAG ACG TAC GTA TAC GTA CGT CTG GGT ACT GGA GAT GTC TTG ATA GCG ACG G 81 O
(output) CAA GAC ATC TCC AGT ACC CCA GTG TT 82 F1 (fuel 1) CAA GAC ATC TCC AGT ACC CAG ACG TAC GTA TAC GTA CG 83 S2 (substrate 2) GGT CTA GGA ATA TAT TCC TAG ACC ACA CCA AAC ACT GGG GTA CTG GAG ATG T 84 T (target-
EGFR del 19) CCG TCG CTA TCA AGA CAT CTC C 85 TNT system P1
(pole 1) ACG GTT GTA CGT ACT AAG CTC AGT CTA TAA ACA CAC CAA ACA CTG GGG TAG ACA CCG GAA CCA AGA CAG CCA CAC GGC GAC AAC AGA AGG TA 86 P2
(pole 2) TAC TTA CCG TGT CTG TGT TGG CCT GTT CCA TCA TAT TTG GCA GGT TTT TCT ACC TTC CGC CGG AAC GCC GGA ACA AGA AGA ACA AGT AGC CG 87 P3 (pole 3) GGA CGA ACT TCA TAT CGC ATG TAC AGG AAT GTT GCT TCT CTT AAT TCC TTC GGC TAC CGG ACC AGC ACC AAG GCG AGA ACA AGA ACG GTG TC -FAM 88 C (cap) TAT GAA GTT CGT CCA TAG ACT GAG CTT AGT ACG TAC AAC CGT GAA CAG GCC AAC ACA GAC ACG GTA AGT ATC CTG TAC ATG CGA 89

<110> Progeneer Inc. <120> SYSTEM FOR DETECTING NUCLEIC ACID <130> P20-B262 <150> KR 2019-0122875 <151> 2019-10-04 <160> 89 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-200b-3p <400> 1 uaauacugcc ugguaaugau ga 22 <210> 2 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-200c <400> 2 uaauacugcc ggguaaugau gga 23 <210> 3 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-let 7 a <400> 3 ugagguagua gguuguauag uu 22 <210> 4 <211 > 22 <212> RNA <213> Artificial Sequence <220> <223> miR-let 7 c <400> 4 ugagguagua gguuguaugg uu 22 <210> 5 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-let 7 d <400> 5 agagguagua gguugcauag uu 22 <210> 6 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-let 7 f <400> 6 ugagguagua gauuguauag uu 22 <210> 7 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-191 <400> 7 caacggaauc ccaaaagcag cug 23 <210> 8 <211> 22 <212> RNA <213 > Artificial Sequence <220> <223> miR-26a <400> 8 uucaaguaa u ccaggauagg cu 22 <210> 9 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-342-3p <400> 9 ucucacacag aaaucgcacc cgu 23 <210> 10 <211> 27 <212 > RNA <213> Artificial Sequence <220> <223> miR-1826 <400> 10 auugaucauc gacacuucga acgcaau 27 <210> 11 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1979 <400> 11 cucccacugc uucacuugac ua 22 <210> 12 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-21 <400> 12 uagcuuauca gacugauguu ga 22 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Output(O)-m 8 toehold <400> 13 cagactgatg ttgaagtacc ccagtgtt 28 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220 > <223> Output(O)-m 10 toehold <400> 14 gactgatgtt gaagtacccc agtgtt 26 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Output(O)-m 12 toehold <400> 15 ctgatgttga agtaccccag tgtt 24 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Fuel 1-No loop 8 stem <400> 16 gactgatgtt gaagtaccga cgtacg 26 <210> 17 < 2 11> 36 <212> DNA <213> Artificial Sequence <220> <223> Fuel 1-loop 8 stem <400> 17 gactgatgtt gaagtaccga cgtacgtata cgtacg 36 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Fuel 1-No loop 10 stem <400> 18 gactgatgtt gaagtaccca gacgtacg 28 <210> 19 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Fuel 1-loop 10 stem <400> 19 gactgatgtt gaagtaccca gacgtacgta tacgtacg 38 <210> 20 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Substrate 1-8 stem <400> 20 gacgtacgta tacgtacgtc ggtacttcaa catcagtctg ataagcta 48 <210> 21 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Substrate 1-10 stem <400> 21 cagacgtacg tatacgtacg tctgggtact tcaacatcag tctgataagc ta 52 <210> 22 <211> 24 <212> DNA <213 > Artificial Sequence <220> <223> Output-F 4 toehold <400> 22 gactgatgtt gaagtaccag tgtt 24 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Output-F 6 toehold <400> 23 gactgatgtt gaagtacccc agtgtt 26 <210> 24 <211> 28 <212> DNA <213> Artificial S equence <220> <223> Output-F 8 toehold <400> 24 gactgatgtt gaagtaccga ccagtgtt 28 <210> 25 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Fuel 1-F 4 toehold < 400> 25 gactgatgtt gaagtacaga cgtacgtata cgtacg 36 <210> 26 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Fuel 1-F 6 toehold <400> 26 gactgatgtt gaagtaccca gacgtacgta tacgtacg 38 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Fuel 1-F 8 toehold <400> 27 gactgatgtt gaagtaccga cagacgtacg tatacgtacg 40 <210> 28 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Substrate 1-F 4 toehold <400> 28 cagacgtacg tatacgtacg tctgtacttc aacatcagtc tgataagcta 50 <210> 29 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Substrate 1-F 6 toehold <400> 29 cagacgtacg tatacgtacg tctgggtact tcaacatcag tctgataagc ta 52 <210> 30 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Substrate 1-F 8 toehold <400> 30 cagacgtacg tatacgtacg tcttgt agtctgataa gcta 54 <210> 31 <211> 52 <212 > DNA <213> Artificial Sequence <220> <223> miR-200b-3p_S1 <400> 31 cagacgtacg tatacgtacg tctgggtact tcatcattac caggcagtat ta 52 <210> 32 <211> 26 <212> DNA <213> Artificial Sequence <220> < 223> miR-200b-3p_O <400> 32 tggtaatgat gaagtacccc agtgtt 26 <210> 33 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> miR-200b-3p_F1 <400> 33 tggtaatgat gaagtaccca gacgtacgta tacgtacg 38 <210> 34 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-200b-3p_S2 <400> 34 ggtctaggaa tatattccta gaccacacca aacactgggg tacttcatca tt 52 <210> 35 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-200c_S1 <400> 35 cagacgtacg tatacgtacg tctgggtact tcagta <ttt 52 > 26 <212> DNA <213> Artificial Sequence <220> <223> miR-200c_O <400> 36 ggtaatgatg gaagtacccc agtgtt 26 <210> 37 <211> 38 <212> DNA <213> Artificial Sequence <220> <223 > miR-200c_F1 <400> 37 ggtaatgatg gaagtaccca gacgtacgta tacgtacg 38 <210> 38 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-200c_S2 <400> 38 ggttctaggaa tatattccta gaccac acca aacactgggg <210> 39 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-let 7a_S1 <400> 39 cagacgtacg tatacgtacg tctgggtact aactatacaa cctactacct ca 52 <210> 40 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> miR-let 7a_O <400> 40 ggttgtatag ttagtacccc agtgtt 26 <210> 41 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> miR-let 7a_F1 <400> 41 ggttgtatag ttagtaccca gacgtacgta t acgtacg 38 <210> 42 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-let 7a_S2 <400> 42 ggtctaggaa tatattccta gaccacacca aacactgggg tactaactat ac 52 <210> 43 <211> 52 <212 > DNA <213> Artificial Sequence <220> <223> miR-let 7c_S1 <400> 43 cagacgtacg tatacgtacg tctgggtact aaccatacaa cctactacct ca 52 <210> 44 <211> 26 <212> DNA <213> Artificial Sequence <220> <223 > miR-let 7c_O <400> 44 ggttgtatgg ttagtacccc agtgtt 26 <210> 45 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> miR-let 7c_F1 <400> 45 ggttgtatgg ttagtaccca gacgtacgta tacgtacg 38 < 210> 46 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-let 7c_S2 <400> 46 ggtctaggaa tatattccta gaccacacca aacactgggg tactaaccat ac 52 <210> 47 <211> 52 <212> DNA < 213> Artificial Sequence <220> <223> miR-let 7d_S1 <400> 47 cagacgtacg tatacgtacg tctgggtact aactatgcaa cctactacct ct 52 <210> 48 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> miR- let 7d_O <400> 48 ggttgcatag ttagtacccc agtgtt 26 <210> 49 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> miR-let 7d_F1 <400> 49 ggttgcatag ttagtaccca gacgtacgta tacgtacg 38 <210> 50 <211> 52 <212> DNA <213> Artificial Sequence <220> < 223> miR-let 7d_S2 <400> 50 ggtctaggaa tatattccta gaccacacca aacactgggg tactaaccat ac 52 <210> 51 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-let 7f_S1 <400> 51 cagacgtacg tatacgtacg tctgggtact aactatacaa tctactacct ca 52 <210> 52 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> miR-let 7f_O <400> 52 gauuguauag uuagtacccc agtgtt 26 <210> 53 <211> 38 <212 > DNA <213> Artificial Sequence <220> <223> miR-let 7f_F1 <400> 53 gauuguauag uuagtaccca gacgtacgta tacgtacg 38 <210> 54 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR -let 7f_S2 <400> 54 ggtctaggaa tatattccta gaccacacca aacactgggg tactaactat ac 52 <210> 55 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-191_S1 <400> 55 cagacgtacg tatacgtacg tctgggtact cagctgcttt tt 52 > 26 <212> DNA <213> Artificial Sequence <220> <223> miR-191_O <400> 56 caaaagcagc tgagtacccc agtgtt 26 <210> 57 <211> 38 <212> DNA <213> Artificial Sequence <220> <223 > miR-191_F1 <400> 57 caaaagcagc tgagtaccca gacgtacgta tacgtacg 38 <210> 58 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-191_S2 <400> 58 ggtctaggaa tatattccta gaccacacca aacactgggg tactcagctgggg <210> 59 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-26a_S1 <400> 59 cagacgtacg tatacgtacg tctgggtact agcctatcct ggattacttg aa 52 <210> 60 <211> 26 <212> DNA < 213> Artificial Sequence <220> <223> miR-26a_O <400> 60 ccaggatagg ctagtacccc agtgtt 26 <210> 61 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> miR-26a_F1 <400> 61 ccaggatagg ctagtaccca gacgtacgta tacgtacg 38 <2 10> 62 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-26a_S2 <400> 62 ggtctaggaa tatattccta gaccacacca aacactgggg tactagccta tc 52 <210> 63 <211> 52 <212> DNA <213 > Artificial Sequence <220> <223> miR-342-3p_S1 <400> 63 cagacgtacg tatacgtacg tctgggtact acgggtgcga tttctgtgtg ag 52 <210> 64 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> miR- 342-3p_O <400> 64 aatcgcaccc gtagtacccc agtgtt 26 <210> 65 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> miR-342-3p_F1 <400> 65 aatcgcaccc gtagtaccca gacgtacgta tacgtacg 38 <210 > 66 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-342-3p_S2 <400> 66 ggtctaggaa tatattccta gaccacacca aacactgggg tactacgggt gc 52 <210> 67 <211> 52 <212> DNA < 213> Artificial Sequence <220> <223> miR-1826_S1 <400> 67 cagacgtacg tatacgtacg tctgggtact attgcgttcg aagtgtcgat ga 52 <210> 68 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> miR-1826_O <400> 68 ttcgaacgca atagtacccc agtgtt 26 <210> 69 <211> 38 <212> DNA <213> A rtificial Sequence <220> <223> miR-1826_F1 <400> 69 ttcgaacgca atagtaccca gacgtacgta tacgtacg 38 <210> 70 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-1826_S2 <400> 70 ggtctaggaa tatattccta gaccacacca aacactgggg tactattgcg tt 52 <210> 71 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-1979_S1 <400> 71 cagacgtacg tatacgtacg tctgggtact tag211 <213> Artificial Sequence <220> <223> miR-1979_S1 <400> 71 cagacgtacg tatacgtacg tctgggtact <211> > 26 <212> DNA <213> Artificial Sequence <220> <223> miR-1979_O <400> 72 ttcacttgac taagtacccc agtgtt 26 <210> 73 <211> 38 <212> DNA <213> Artificial Sequence <220> <223 > miR-1979_F1 <400> 73 ttcacttgac taagtaccca gacgtacgta tacgtacg 38 <210> 74 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-1979_S2 <400> 74 ggtctaggaa tatattccta gaccacacca aacactgggg tacttagtca ag 52 <210> 75 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-21_S1 <400> 75 cagacgtacg tatacgtacg tctgggtact tcaacatcag tctga < 76211> 52 <212> > 26 <212> DNA <213> Artificial Sequence <220> <223> miR-21_O <400> 76 gactgatgtt gaagtacccc agtgtt 26 <210> 77 <211> 38 <212> DNA <213> Artificial Sequence <220> <223 > miR-21_F1 <400> 77 gactgatgtt gaagtaccca gacgtacgta tacgtacg 38 <210> 78 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> miR-21_S2 <400> 78 ggtctaggaa tatattccta gaccac acca aacactgg gg <210> 79 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Universal_U <400> 79 taccccagtg tttggtgtgt tt 22 <210> 80 <211> 39 <212> DNA <213> Artificial Sequence < 220> <223> Universal_F2 <400> 80 taccccagtg tttggtgtgg tctaggaata tattcctag 39 <210> 81 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> EGFR del 19-S1 <400> 81 cagacgtacg tatacgtacg tctgggtact ggagatgtct tgatagcgac gg 52 < 210> 82 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> EGFR del 19-O <400> 82 caagacatct ccagtacccc agtgtt 26 <210> 83 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> EGFR del 19-F1 <400> 83 caagacatct ccagtaccca gacgtacgta tacgtacg 38 <210> 84 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> EGFR del 19-S2 <400> 84 ggtctaggaa tatattccta gaccacacca aacactgggg tactggagat gt 52 <210> 85 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> EGFR del 19-Target <400> 85 ccgtcgctat caagacatct cc 22 <210> 86 <211> 92 <212> DNA <213> Artificial Sequence <220> <223> TNT-P1 <400> 86 acggttgtac gtactaagct cagtctataa acacaccaaa cactggggta gacaccggaa 60 ccaagacagc cacacggcga caacagaagg ta 92 <210> 92 <212> DNA <211> 92 <212> DNA <213> Artificial Sequence <220> <223> TNT-P1 <400> 86 Artificial Sequence <220> <223> TNT-P2 <400> 87 tacttaccgt gtctgtgttg gcctgttcca tcatatttgg caggtttttc taccttccgc 60 cggaacgccg gaacaagaag aacaagtagc cg 92 <210> 88 <211> < 92 <220> Artificial Sequence <220> DNA TNT-P3 <400> 88 ggacgaactt catatcgcat gtacaggaat gttgcttctc ttaattcctt cggctaccgg 60 accagcacca aggcgagaac aagaacggtg tc 92 <210> 89 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> T NT gtccatagac tgagcttagt acgtacaacc gtgaacaggc caacacagac 60acggtaagta tcctgtacat gcga 84

Claims (29)

1) sequentially comprising a nucleic acid sequence A1, a nucleic acid sequence A2, a first toehold sequence A3, a nucleic acid sequence A4 and a second toehold sequence A5, wherein the nucleic acid sequence A4 and the second toehold sequence A5 as a whole are all of the target nucleic acid or a first nucleic acid S1 (Substrat 1) that is a complementary sequence capable of binding to a partial sequence, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure;
2) a second nucleic acid O (Output) sequentially comprising a sequence complementary to the nucleic acid sequence A4 and a sequence complementary to the first toehold sequence A3;
3) a third nucleic acid F1 (Fuel 1) sequentially comprising a sequence complementary to a part of the nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or part of the nucleic acid sequence A2;
4) sequentially comprising a nucleic acid sequence B1, a nucleic acid sequence B2, a third toehold sequence B3, a nucleic acid sequence B4 and a fourth toehold sequence B5, wherein the nucleic acid sequence B4 and the fourth toehold sequence B5 as a whole are the second nucleic acid O a fourth nucleic acid S2 (Substrate 2), which is a complementary sequence capable of binding to all or part of a sequence, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure;
5) a fifth nucleic acid U (Universal code) sequentially comprising a sequence complementary to the nucleic acid sequence B4 and a sequence complementary to the third toehold sequence B3; and
6) a sixth nucleic acid F2 (Fuel 2) comprising sequentially a sequence complementary to a part of the nucleic acid sequence B4, a sequence complementary to the third toehold sequence B3, and a sequence complementary to all or a part of the nucleic acid sequence B2; A composition for detecting a target nucleic acid.
The method of claim 1, wherein the second nucleic acid O and the first nucleic acid S1 complementarily bind to form a double strand (S10), and complementarily bind with the fifth nucleic acid U and the fourth nucleic acid S2 to form a double strand (S2U) ) forming a composition for detecting a target nucleic acid.
The composition for detecting a target nucleic acid according to claim 1, wherein the second toehold sequence A5 and the fourth toehold sequence B5 are exposed as single strands.
The composition for detecting a target nucleic acid according to claim 1, wherein the second toehold sequence A5 or the fourth toehold sequence B5 comprises 4 to 8 nucleotides.
The composition for detecting a target nucleic acid according to claim 1, wherein the third nucleic acid F1 or the sixth nucleic acid F2 has a hairpin structure.
The composition for detecting a target nucleic acid according to claim 5, wherein the stem portion of the third nucleic acid F1 or the sixth nucleic acid F2 contains 6 to 12 nucleotides.
The composition for detecting a target nucleic acid according to claim 1, wherein the first toehold sequence A3 or the third toehold sequence B3 comprises 4 to 12 nucleotides.
The method according to claim 1, wherein when the target nucleic acid is complementary to the second toehold sequence A5 of the first nucleic acid S1, the second nucleic acid O is released through a branch migration reaction, and the first toehold sequence A3 is exposed, a composition for detecting a target nucleic acid.
The composition for detecting a target nucleic acid according to claim 8, wherein the first toehold sequence A3 of the first nucleic acid S1 bound to the target nucleic acid and the third nucleic acid F1 bind to release the target nucleic acid through a branch point shift reaction.
10. The method according to claim 9, wherein the released target nucleic acid is re-reacted with the second toehold sequence A5 of the second nucleic acid O and the first nucleic acid S1 (S10) that are complementary to form a double strand. composition.
The composition for detecting a target nucleic acid according to claim 8, wherein the released second nucleic acid O binds complementary to the fourth toehold sequence B5 of the fourth nucleic acid S2 to release the fifth nucleic acid U through a branch point shift reaction.
The composition for detecting a target nucleic acid according to claim 11, wherein the third toehold sequence B3 of the fourth nucleic acid S2 bound to the second nucleic acid O binds to the sixth nucleic acid F2 to release the second nucleic acid O through a branch point shift reaction. .
13. The target nucleic acid according to claim 12, wherein the released second nucleic acid O is re-reacted with the fourth toehold sequence B5 of the fourth nucleic acid S2 and the fifth nucleic acid U (S2U), which are complementarily bound to form a double strand. composition for detection.
The composition for detecting a target nucleic acid according to claim 1, wherein the detection signal of the target nucleic acid is amplified as the fifth nucleic acid U released through a toehold mediated strand displacement reaction.
The composition for detecting a target nucleic acid according to claim 14, wherein the detection signal of the target nucleic acid is amplified by self-assembly of the ineffective element.
The composition for detecting a target nucleic acid according to claim 1, wherein the first nucleic acid S1 further comprises a quencher.
17. The method of claim 16, wherein the quencher is Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa black FQ, Iowa black RQ, at least one selected from the group IRDye QC-1, a composition for detecting a target nucleic acid.
The composition for detecting a target nucleic acid according to claim 1, wherein the second nucleic acid O and the fifth nucleic acid U contain a fluorescent substance.
The method of claim 18, wherein the fluorescent material is fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, or tetramethylrhodamine. , FITC, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, Cyanine-based dyes and Ciadicarbosy At least one selected from the group consisting of thiadicarbocyanine, a composition for detecting a target nucleic acid.
The composition for detecting a target nucleic acid according to claim 1, further comprising a fluorescent nucleic acid nanostructure comprising a single-stranded probe comprising a nucleic acid complementary to the fifth nucleic acid U and a fluorescent substance, and graphene oxide.
1) sequentially comprising a nucleic acid sequence A1, a nucleic acid sequence A2, a first toehold sequence A3, a nucleic acid sequence A4 and a second toehold sequence A5, wherein the nucleic acid sequence A4 and the second toehold sequence A5 as a whole are all of the target nucleic acid or a first nucleic acid S1 that is a complementary sequence capable of binding to a partial sequence, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure;
2) a second nucleic acid O sequentially comprising a sequence complementary to the nucleic acid sequence A4 and a sequence complementary to the first toehold sequence A3;
3) a third nucleic acid F1 sequentially comprising a sequence complementary to a portion of the nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or a portion of the nucleic acid sequence A2;
4) sequentially comprising a nucleic acid sequence B1, a nucleic acid sequence B2, a third toehold sequence B3, a nucleic acid sequence B4 and a fourth toehold sequence B5, wherein the nucleic acid sequence B4 and the fourth toehold sequence B5 as a whole are the second nucleic acid O a fourth nucleic acid S2 that is a complementary sequence capable of binding to all or part of a sequence, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure;
5) a fifth nucleic acid U sequentially comprising a sequence complementary to nucleic acid sequence B4 and a sequence complementary to a third toehold sequence B3; and
6) a target nucleic acid comprising a sixth nucleic acid F2 sequentially comprising a sequence complementary to a portion of nucleic acid sequence B4, a sequence complementary to a third toehold sequence B3 and a sequence complementary to all or a portion of nucleic acid sequence B2 detection kit.
1) sequentially comprising a nucleic acid sequence A1, a nucleic acid sequence A2, a first toehold sequence A3, a nucleic acid sequence A4 and a second toehold sequence A5, wherein the nucleic acid sequence A4 and the second toehold sequence A5 as a whole are all of the target nucleic acid or a first nucleic acid S1 that is a complementary sequence capable of binding to a partial sequence, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure;
2) a second nucleic acid O sequentially comprising a sequence complementary to the nucleic acid sequence A4 and a sequence complementary to the first toehold sequence A3;
3) a third nucleic acid F1 sequentially comprising a sequence complementary to a portion of the nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or a portion of the nucleic acid sequence A2;
4) sequentially comprising a nucleic acid sequence B1, a nucleic acid sequence B2, a third toehold sequence B3, a nucleic acid sequence B4 and a fourth toehold sequence B5, wherein the nucleic acid sequence B4 and the fourth toehold sequence B5 as a whole are the second nucleic acid O a fourth nucleic acid S2 that is a complementary sequence capable of binding to all or part of a sequence, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure;
5) a fifth nucleic acid U sequentially comprising a sequence complementary to nucleic acid sequence B4 and a sequence complementary to a third toehold sequence B3; and
6) a sixth nucleic acid F2 comprising sequentially a sequence complementary to a portion of nucleic acid sequence B4, a sequence complementary to a third toehold sequence B3, and a sequence complementary to all or a portion of nucleic acid sequence B2; A composition for universal code conversion, which converts a target nucleic acid into a fifth nucleic acid U, which is a predetermined universal code, through a mediated strand displacement reaction.
The composition for universal code conversion according to claim 22, wherein the fourth nucleic acid sequence B5 of the first nucleic acid S1, the second nucleic acid O, the third nucleic acid F1 and the fourth nucleic acid S2 is changed according to the target nucleic acid.
The composition for universal code conversion according to claim 22, wherein the sixth nucleic acid F2 and the fifth nucleic acid U are fixed to a predetermined sequence regardless of the target nucleic acid.
1) sequentially comprising a nucleic acid sequence A1, a nucleic acid sequence A2, a first toehold sequence A3, a nucleic acid sequence A4 and a second toehold sequence A5, wherein the nucleic acid sequence A4 and the second toehold sequence A5 as a whole are all of the target nucleic acid or a first nucleic acid S1 that is a complementary sequence capable of binding to a partial sequence, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure;
2) a second nucleic acid O sequentially comprising a sequence complementary to the nucleic acid sequence A4 and a sequence complementary to the first toehold sequence A3;
3) a third nucleic acid F1 sequentially comprising a sequence complementary to a portion of the nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or a portion of the nucleic acid sequence A2;
4) sequentially comprising a nucleic acid sequence B1, a nucleic acid sequence B2, a third toehold sequence B3, a nucleic acid sequence B4 and a fourth toehold sequence B5, wherein the nucleic acid sequence B4 and the fourth toehold sequence B5 as a whole are the second nucleic acid O a fourth nucleic acid S2 that is a complementary sequence capable of binding to all or part of a sequence, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure;
5) a fifth nucleic acid U sequentially comprising a sequence complementary to nucleic acid sequence B4 and a sequence complementary to a third toehold sequence B3; and
6) a sixth nucleic acid F2 comprising sequentially a sequence complementary to a portion of nucleic acid sequence B4, a sequence complementary to a third toehold sequence B3, and a sequence complementary to all or a portion of nucleic acid sequence B2,
A universal code conversion kit for converting a target nucleic acid into a fifth nucleic acid U, which is a predetermined universal code, through a toehold-mediated strand displacement reaction.
1) In the kit for detecting a target nucleic acid according to claim 21, the nucleic acid sequence A4 and the second toehold sequence A5 of the first nucleic acid S1 are changed to a complementary sequence capable of binding to all or part of the target nucleic acid sequence, and thus the second nucleic acid O; altering the fourth toehold sequence B5 of the third nucleic acid F1 and the fourth nucleic acid S2; and
2) A method of detecting a target nucleic acid, comprising detecting a fifth nucleic acid U released via a toehold mediated strand displacement reaction.
The method of claim 26, wherein the released fifth nucleic acid U is detected using a fluorescent nucleic acid nanostructure specific for the released fifth nucleic acid U and graphene oxide.
a hybrid of a third nucleic acid F1 and a sixth nucleic acid F2;
a double strand (S10) to which the second nucleic acid O and the first nucleic acid S1 are complementary; and
A biosensor for detecting a target nucleic acid comprising a double strand (S2U) complementary to a fifth nucleic acid U and a fourth nucleic acid S2,
wherein the first nucleic acid S1 sequentially comprises a nucleic acid sequence A1, a nucleic acid sequence A2, a first threshold sequence A3, a nucleic acid sequence A4 and a second threshold sequence A5, wherein the nucleic acid sequence A4 and the second threshold sequence A5 as a whole a complementary sequence capable of binding to all or part of a sequence of a target nucleic acid, wherein the nucleic acid sequence A1 and the nucleic acid sequence A2 are complementary to each other to form a hairpin structure;
said second nucleic acid O sequentially comprises a sequence complementary to nucleic acid sequence A4 and a sequence complementary to first toehold sequence A3;
the third nucleic acid F1 sequentially comprises a sequence complementary to a part of nucleic acid sequence A4, a sequence complementary to the first toehold sequence A3, and a sequence complementary to all or part of the nucleic acid sequence A2;
wherein the fourth nucleic acid S2 sequentially comprises a nucleic acid sequence B1, a nucleic acid sequence B2, a third threshold sequence B3, a nucleic acid sequence B4 and a fourth threshold sequence B5, wherein the nucleic acid sequence B4 and the fourth threshold sequence B5 as a whole a complementary sequence capable of binding to all or part of the sequence of the second nucleic acid O, wherein the nucleic acid sequence B1 and the nucleic acid sequence B2 are complementary to each other to form a hairpin structure;
the fifth nucleic acid U sequentially comprises a sequence complementary to the nucleic acid sequence B4 and a sequence complementary to the third toehold sequence B3;
The sixth nucleic acid F2 is a sequence complementary to a portion of the nucleic acid sequence B4, a sequence complementary to the third toehold sequence B3, and a sequence complementary to all or a portion of the nucleic acid sequence B2. Target nucleic acid detection bio sensor.
The biosensor for detecting a target nucleic acid according to claim 28, further comprising a fluorescent nucleic acid nanostructure comprising a single-stranded probe comprising a nucleic acid complementary to the fifth nucleic acid U and a fluorescent substance, and graphene oxide.

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