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WO2021075764A1 - Procédé de détection de multiples biomarqueurs - Google Patents

Procédé de détection de multiples biomarqueurs Download PDF

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WO2021075764A1
WO2021075764A1 PCT/KR2020/013235 KR2020013235W WO2021075764A1 WO 2021075764 A1 WO2021075764 A1 WO 2021075764A1 KR 2020013235 W KR2020013235 W KR 2020013235W WO 2021075764 A1 WO2021075764 A1 WO 2021075764A1
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nucleic acid
acid sequence
biomarker
complementary
target
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Korean (ko)
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엄숭호
육지수
김정훈
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Progeneer Inc
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/131—Modifications characterised by incorporating a restriction site
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/143—Magnetism, e.g. magnetic label
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/149—Particles, e.g. beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/10—Detection mode being characterised by the assay principle
    • C12Q2565/101—Interaction between at least two labels

Definitions

  • the present invention relates to a method for detecting multiple biomarkers, and more particularly, to a method for improving detection of multiple biomarkers using a fluorescent nucleic acid nanostructure-graphene oxide complex through a nucleic acid pretreatment process.
  • a method of detecting a specific nucleic acid (DNA or RNA) or protein is a fundamentally important technology in the field of scientific research. By being able to detect and identify specific nucleic acids or proteins, researchers can determine which genetic and biological markers are indicative of a person's health status. By using such a method of detecting nucleic acids and proteins, it is possible to detect modifications of pathogen genes present in a sample or expression of specific genes.
  • 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.
  • As a representative molecular diagnostic technology there is a PCR technology that amplifies DNA within a short time (Saiki, R., et. al.
  • Cepheid's GeneXprt system and reagents which are PCR products with a field diagnosis concept, have been developed and sold, but it is difficult to use them in general tests because equipment and reagents are very expensive (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).
  • there is a Nucleic Acid Lateral Flow Assay which uses a membrane instead of gel electrophoresis after PCR to confirm (Aveyard, J., et.
  • cancer diagnosis technique there are various methods such as molecular biological diagnosis, ultrasound diagnosis, X-ray, MRI, CT, PET, and bone scan. Since most cancer diagnosis techniques are finally judged through the naked eye, a large error may occur in the process of reading radiographic films or tissues. That is, depending on the size of the cancer, it can be diagnosed by mistaken detection of cancer or simple inflammation. 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 the diagnosis. In addition, the risk of radiation exposure to patients is an inevitable problem.
  • the tumor tissue may be directly collected and tested through biopsy, but in this case, only a portion of the suspected cancer tissue is examined, so the overall pattern and type of cancer cannot be grasped. .
  • the use of needles or surgical therapies to collect tumors can also give patients a great sense of rejection, and there are cases in which tissue biopsy itself is not possible depending on the patient's condition.
  • the existing methods used to confirm cancer are expensive and inconvenient to patients, so they are not suitable for periodic and frequent examinations.
  • attempts are being made to diagnose cancer by detecting biomarkers in the patient's blood, urine, and saliva through a liquid biopsy.
  • sncRNAs short non-coding RNAs
  • miRNAs RNA and DNA interacting with each other
  • studies on the correlation between sncRNA profiling and genetic mutations of genes are being conducted, but there has been a problem in designing each probe according to various short sequences that are noted in sequencing analysis. Therefore, it is very essential to develop a method for simultaneously diagnosing short and long sequences.
  • An object of the present invention is to provide a method for detecting a biomarker.
  • an object of the present invention is to provide a biomarker detection kit.
  • the present invention comprises the steps of separating a nucleic acid from a sample; Selecting a target nucleic acid in the biomarker by pretreating the nucleic acid; Preparing a fluorescent nucleic acid nanostructure-graphene oxide complex; And detecting a target nucleic acid.
  • the present invention includes a restriction enzyme recognition sequence, a forward primer specifically binding to a target nucleic acid in a biomarker, and a reverse primer in which biotin is bound to the 5'end; Restriction enzymes; Magnetic particles coated with streptavidin; Fluorescent nucleic acid nanostructure; And it provides a biomarker detection kit comprising a pin oxide.
  • FIG. 1 is a diagram schematically showing a method for detecting a biomarker of the present invention.
  • Step 1 Separating the nucleic acid from the sample
  • Step 2 and 3 Pretreatment of nucleic acid to select target nucleic acid
  • Step 4 preparing a fluorescent nucleic acid nanostructure-graphene oxide complex and detecting a target nucleic acid
  • FIG. 2 is a diagram showing a developed view model of a triangular columnar fluorescent nucleic acid nanostructure combined with a target sequence:
  • c1, c2 and c3 target sequence.
  • FIG. 3 is a diagram showing the Cy5/Cy3 fluorescence values for each NaCl concentration condition in the heat treatment process during the manufacturing process of the triangular columnar fluorescent nucleic acid nanostructure.
  • FIG. 4 is a diagram showing normalized Cy5 and Cy3 fluorescence values according to NaCl concentration conditions in a heat treatment process during a manufacturing process of a triangular columnar fluorescent nucleic acid nanostructure.
  • FIG. 5 is a diagram showing an exploded view model of a tetrahedral fluorescent nucleic acid nanostructure combined with a target sequence.
  • FIG. 6 is a diagram showing a normalized fluorescence value according to the concentration of a target sequence.
  • FIG. 7 is a table showing a trend line equation of the fluorescence values measured in FIG. 6 and a detection limit value obtained at this time.
  • LW L858R wild type
  • TW T790M wild type
  • FIG. 9 is a diagram showing changes in normalized fluorescence values and Cy5/Cy3 fluorescence values according to the ratio of wild type and mutation.
  • FIG. 10 is a diagram showing the calculation of the minimum mutation rate that can be identified in a 200nM target using the Cy5/Cy3 fluorescence values measured in FIG. 11.
  • FIG. 11 is a diagram showing the Cy5/Cy3 fluorescence ratio in the triangular columnar fluorescent nucleic acid nanostructure prepared according to the length of the target sequence (Del Ex19).
  • Green represents a wild-type Del Ex19
  • red represents a Cy5/Cy3 ratio when targeting a Del Ex19 mutation.
  • 11B shows the system efficiency ( ⁇ (%)) calculated using the following equation.
  • FIG. 12 is a diagram showing the fluorescence ratio of Cy5/Cy3 in a triangular columnar fluorescent nucleic acid nanostructure prepared according to the length of a single-stranded (a) or double-stranded (b) target nucleic acid.
  • FIG. 13 is a diagram showing the detection ability of Del Ex19 targets for RNA samples extracted from four types of lung cancer cell lines of triangular columnar fluorescent nucleic acid nanostructures prepared according to the length of the target sequence (Del Ex19):
  • P+R+F PCR, restriction enzyme reaction, and filtering process using magnetic particles.
  • L858R target detection ability for RNA samples extracted from four types of lung cancer cell lines of a triangular columnar fluorescent nucleic acid nanostructure prepared according to the length of the target sequence (L858R).
  • 15 is a schematic diagram showing the sequence relationship of the triangular columnar fluorescent nucleic acid nanostructure of the present invention.
  • 16 is a schematic diagram showing the sequence relationship of the tetrahedral fluorescent nucleic acid nanostructure of the present invention.
  • nucleic acids are written in a 5' ⁇ 3' orientation from left to right.
  • Numerical ranges recited within the specification include the numbers defining the range, and include each integer or any non-integer fraction within the defined range.
  • the present invention provides a step of amplifying a nucleic acid obtained by separating a target nucleic acid region including a position where a nucleic acid variant occurs in a biomarker with a forward primer and a reverse primer in which biotin is bound to the 5'end; And it relates to a biomarker pretreatment method comprising the step of treating the amplified product with a restriction enzyme and treating magnetic particles coated with streptavidin to obtain a target nucleic acid of the biomarker.
  • a long-length biomarker nucleic acid sequence may be converted into a short-length target nucleic acid including a location where a nucleic acid variant occurs and selected.
  • the transformed short-length target nucleic acid may be characterized as being less than 75 bp in length, more preferably 5 to 50 bp in length.
  • the converted short-length target nucleic acid when it is a single strand, it may be preferably 43 bp or less in length, and more preferably 16-43 bp in length.
  • the converted short-length target nucleic acid when the converted short-length target nucleic acid is double-stranded, it may be preferably 28 bp or less in length, and more preferably 16-28 bp.
  • the primer may include a restriction enzyme site.
  • the target portion may be cut to be 20 to 30 bp.
  • the present invention comprises the steps of separating a nucleic acid from a sample; Selecting a target nucleic acid of a biomarker by pretreating the nucleic acid; Preparing a fluorescent nucleic acid nanostructure-graphene oxide complex; And detecting a target nucleic acid.
  • the pretreatment of the nucleic acid comprises the steps of amplifying a nucleic acid obtained by separating a target nucleic acid region including a position where a nucleic acid variant occurs in a biomarker with a forward primer and a reverse primer conjugated with biotin at the 5'end; And treating the amplified product with a restriction enzyme and treating magnetic particles coated with streptavidin to obtain a target nucleic acid of the biomarker, wherein the primer is a restriction enzyme recognition sequence. site).
  • the detection method of the present invention may multiplely detect one or more biomarkers.
  • the fluorescent nucleic acid nanostructure may have a triangular column shape or a tetrahedral shape.
  • the triangular columnar fluorescent nucleic acid nanostructure comprises: a first nucleic acid U1 consisting of a nucleic acid sequence A1, a nucleic acid sequence A2, a nucleic acid sequence A3a, and a nucleic acid sequence A3b;
  • a second nucleic acid S1 sequentially comprising a first fluorescent substance, a nucleic acid sequence complementary to a nucleic acid sequence A1, a nucleic acid sequence complementary to a target nucleic acid c1 of the first biomarker, a nucleic acid sequence B1, a nucleic acid sequence B2, and a nucleic acid sequence B3;
  • a second fluorescent substance a nucleic acid sequence complementary to a nucleic acid sequence A2, a nucleic acid sequence complementary to a target nucleic acid c2 of a second biomarker, a nucleic acid sequence complementary to a nucleic acid sequence B3, a nucleic acid sequence B4, and a nucleic acid sequence B5 in sequence 3 nucle
  • the tetrahedral fluorescent nucleic acid nanostructure comprises: a first nucleic acid U1 consisting of a nucleic acid sequence A1, a nucleic acid sequence A2, and a nucleic acid sequence A3;
  • a second nucleic acid S1 sequentially comprising a first fluorescent substance, a nucleic acid sequence B1, a nucleic acid sequence complementary to a nucleic acid sequence A1, a nucleic acid sequence B2, a nucleic acid sequence complementary to a target nucleic acid c1 of the first biomarker, and a nucleic acid sequence B3;
  • a second fluorescent substance a nucleic acid sequence B4, a nucleic acid sequence complementary to a nucleic acid sequence A2, a nucleic acid sequence complementary to a nucleic acid sequence B1, a nucleic acid sequence complementary to a target nucleic acid c2 of the second biomarker, and a nucleic acid sequence B5 in sequence.
  • nucleic acid sequence B2 a nucleic acid sequence complementary to a nucleic acid sequence A3, a nucleic acid sequence complementary to a nucleic acid sequence B4, a nucleic acid sequence complementary to a target nucleic acid c3 of a third biomarker, a nucleic acid sequence B6. It may include a fourth nucleic acid S3 comprising sequentially.
  • Biomarkers Del Ex19, T790M, and L858R used as target sequences in an embodiment of the present invention are all genetic mutation markers occurring in the epidermal growth factor receptor (EGFR) gene.
  • EGFR epidermal growth factor receptor
  • Del Ex19 is a deletion mutation occurring in Exon 19 of the EGFR gene
  • T790M is a point mutation occurring in Exon 20
  • L858R is a point mutation occurring in Exon 21.
  • These markers are known as mutations sensitive to the drug EGFR TKI (Epidermal growth factor receptor tyrosine kinase inhibitor), and are used as reference points for drug prescription related to EGFR TKI.
  • the fluorescent nucleic acid nanostructure-graphene oxide complex is heat treated after mixing the target nucleic acid selected through the pretreatment of the present invention, the first nucleic acid U1, the second nucleic acid S1, the third nucleic acid S2, and the fourth nucleic acid S3.
  • it can be prepared by preparing a fluorescent nucleic acid nanostructure and then attaching it to graphene oxide.
  • the fluorescent nanonucleic acid structure of the present invention is complementary to the targets of S1, S2 and S3 in the structure when there is no target nucleic acid (sequence), that is, c1, c2 and c3 in the case of a triangular columnar fluorescent nanonucleic acid structure.
  • All three-dimensional structures are formed by junctions that form double bonds except for the partial (c1*, c2*, and c3*) and one side of the triangular bottom surface of the triangular prism bonded to the graphene oxide;
  • fluorescent nanonucleic acid structures including target nucleic acids c1, c2 and c3 are prepared or reacted to form fluorescent nanonucleic acid structures
  • target nucleic acids c1, c2 and c3 are c1*, c2* and c3* of S1, S2 and S3.
  • a double bond is formed on all but one side of the triangular surface of the triangular prism to be attached to the graphene oxide.
  • a target nucleic acid that is, c1, c2, and c3, portions complementary to the targets of S1, S2 and S3 in the structure (c1*, c2* and c3*) and yes Except for the poly A tail portion (B3, B5 and B6) to be attached to the pin oxide, all form a double bond to form a three-dimensional structure (junction);
  • the target nucleic acids c1, c2 and c3 are a single-stranded sequence portion complementary to each of S1, S2 and S3. Since it binds to, all forms a double bond except for the poly A tail to be attached to the graphene oxide.
  • detection of the target nucleic acid may be detected by detecting fluorescence with a fluorescence resonance energy transfer phenomenon.
  • the nucleic acid can be DNA and/or RNA.
  • fluorescent nanonucleic acid structure includes a single-stranded probe comprising a nucleic acid and a fluorescent material having a sequence complementary to a target nucleic acid, a structure consisting of the probes, or the probes bound to the target nucleic acid.
  • 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.
  • the term "probe” refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases or hundreds of bases that can achieve specific binding to mRNA, and is labeled to confirm the presence or absence of a specific nucleic acid.
  • 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, etc., but in the present invention, it means a single-stranded nucleic acid. It may be one nucleic acid U1, a second nucleic acid S1, a third nucleic acid S2, and a fourth nucleic acid S3.
  • sample includes tissue, cells, blood, serum, urine, saliva, plasma or body fluid obtained from a subject or patient, and the source of the tissue or cell sample is fresh, frozen and/or Solid tissue from preserved organ or tissue samples or biopsies or aspirates; Blood or any blood component; It may be a cell at any point in the subject's pregnancy or development.
  • the tissue sample may also be a primary or cultured cell or cell line, more preferably a liquid biopsy.
  • the term “detection” or “measurement” means quantifying the concentration of a detected or measured object.
  • it may include one or more single-stranded probes comprising a nucleic acid complementary to a target nucleic acid, and if three or more single-stranded probes comprising a nucleic acid complementary to a target nucleic acid are present, the target nucleic acid of each single-stranded probe
  • a three-dimensional structure can be achieved by additionally including a single-stranded probe consisting of sequences complementary to some sequences excluding a nucleic acid site complementary to.
  • multiple detections of different target nucleic acids are possible by including one or more types of single-stranded probes containing nucleic acids complementary to different target nucleic acids and different fluorescent substances, respectively, and stable detection results are maintained by forming a three-dimensional structure. It has the advantage of being able to quantify several target nucleic acids, respectively.
  • nucleic acids specific for a specific disease may be selected as targets and used for diagnosis thereof.
  • the target sequence is pretreated to include the mutations Del Ex19, L858R, and T790M known as lung cancer-specific biomarkers to make short-length nucleic acids, and then used as c1, respectively, and of the same length without mutations. Since the nucleic acid was used as c3 and GAPDH was used as c2, the diagnosis of lung cancer and the effect of using anticancer drugs can be monitored using this.
  • the probe of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well known methods.
  • Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoro Amidates, carbamates, etc.) or to charged linkers (eg phosphorothioate, phosphorodithioate, etc.).
  • uncharged linkers e.g., methyl phosphonate, phosphotriester, phosphoro Amidates, carbamates, etc.
  • charged linkers eg phosphorothioate, phosphorodithioate, etc.
  • the second nucleic acid S1, the second nucleic acid S1, the third nucleic acid S2, and the fourth nucleic acid S3, and a single-stranded probe containing different fluorescent substances from the target nucleic acid selected through the pretreatment of the present invention U1 can be assembled into a fluorescent nanonucleic acid structure through a specific heat treatment process.
  • the fluorescent materials included in the single-stranded probes may be fluorescent materials of different wavelength bands.
  • the fluorescent material is fluorescein (FAM), Texas Red, rhodamine, Alexa, cyanine (Cy), BODIPY, acetoxymethyl It may be selected from ester (Acetoxymethyl ester) and coumarin (coumarin), but is not limited thereto, and any known phosphor capable of attaching a single strand of nucleic acid may be used.
  • the heat treatment may be different depending on the structure of the fluorescent nanonucleic acid structure, and in the case of a tetrahedron, 95° C., 2 minutes; Cooling at 60° C. at 1° C. per minute; Can be heat-treated at 20°C for 5 minutes and 4°C, and for triangular pillars, 95°C for 5 minutes; 85° C. for 5 minutes; It can be heat-treated at 20°C for 5 minutes and 4°C by lowering 0.5°C per minute.
  • graphene oxide is a fluorescence limiting chemical (Universal Quencher: UQ) and can selectively block fluorescence due to fluorescence resonance energy transfer, and the target nucleic acid and the single-stranded probe of the present invention are When combined to form a double bond, the probe (or fluorescent nanonucleic acid structure) is farther away from the graphene oxide, so that light emission of the fluorescent material can be detected.
  • UQ fluorescence limiting chemical
  • graphene oxide is more easily attached to a single-stranded base sequence than a double-stranded base sequence, and the distance between the graphene oxide and the fluorescent material of the fluorescent nanonucleic acid structure according to the fluorescence resonance energy transfer Fluorescence is blocked when the distance is increased, and when the distance is increased, multiple target nucleotide sequences are combined with a sequence complementary to each of the probes, using the property that fluorescence can be detected again.
  • the complex was designed to be able to recognize and quantify multiple targets at once by emitting fluorescence as the distance from the graphene oxide was increased when.
  • the concentration of the salt (NaCl) in the step of preparing the fluorescent nanonucleic acid structure and attaching the fluorescent nanonucleic acid structure to the graphene oxide may be 50 to 300 mM.
  • the present invention comprises the steps of separating a nucleic acid from a sample; Selecting a target nucleic acid of a biomarker by pretreating the nucleic acid; Preparing a fluorescent nucleic acid nanostructure-graphene oxide complex; And reacting the selected target nucleic acid with the fluorescent nucleic acid nanostructure-graphene oxide complex to detect the target nucleic acid.
  • the pretreatment of the nucleic acid comprises the steps of amplifying a nucleic acid obtained by separating a target nucleic acid region including a position where a nucleic acid variant occurs in a biomarker with a forward primer and a reverse primer conjugated with biotin at the 5'end; And treating the amplified product with a restriction enzyme, and treating magnetic particles coated with streptavidin to obtain a target nucleic acid of the biomarker, wherein the primer includes a restriction enzyme recognition sequence.
  • the primer includes a restriction enzyme recognition sequence.
  • the fluorescent nucleic acid nanostructure of the target nucleic acid and the fluorescent nucleic acid nanostructure-graphene oxide complex reacts, a double bond is formed and the distance from the graphene oxide increases, so that light emission of the fluorescent material may be detected.
  • the fluorescent nucleic acid nanostructure may have a triangular column shape or a tetrahedral shape.
  • the triangular columnar fluorescent nucleic acid nanostructure comprises: a first nucleic acid U1 consisting of a nucleic acid sequence A1, a nucleic acid sequence A2, and a nucleic acid sequence A3;
  • a second nucleic acid S1 sequentially comprising a first fluorescent substance, a nucleic acid sequence complementary to a nucleic acid sequence A1, a nucleic acid sequence complementary to a target nucleic acid c1 of the first biomarker, a nucleic acid sequence B1, a nucleic acid sequence B2, and a nucleic acid sequence B2;
  • a second fluorescent substance a nucleic acid sequence complementary to a nucleic acid sequence A2, a nucleic acid sequence complementary to a target nucleic acid c2 of a second biomarker, a nucleic acid sequence complementary to a nucleic acid sequence B2, a nucleic acid sequence B4, and a nucleic acid sequence B5 in sequence 3 nucleic acid S2;
  • the tetrahedral fluorescent nucleic acid nanostructure comprises: a first nucleic acid U1 consisting of a nucleic acid sequence A1, a nucleic acid sequence A2, and a nucleic acid sequence A3;
  • a second nucleic acid S1 sequentially comprising a first fluorescent substance, a nucleic acid sequence B1, a nucleic acid sequence complementary to a nucleic acid sequence A1, a nucleic acid sequence B2, a nucleic acid sequence complementary to a target nucleic acid c1 of the first biomarker, and a nucleic acid sequence B3;
  • a second fluorescent substance a nucleic acid sequence B4, a nucleic acid sequence complementary to a nucleic acid sequence A2, a nucleic acid sequence complementary to a nucleic acid sequence B1, a nucleic acid sequence complementary to a target nucleic acid c2 of the second biomarker, and a nucleic acid sequence B5 in sequence.
  • nucleic acid sequence B2 a nucleic acid sequence complementary to a nucleic acid sequence A3, a nucleic acid sequence complementary to a nucleic acid sequence B4, a nucleic acid sequence complementary to a target nucleic acid c3 of a third biomarker, a nucleic acid sequence B6. It may include a fourth nucleic acid S3 comprising sequentially.
  • the present invention comprises a restriction enzyme recognition sequence, a forward primer specifically binding to the target nucleic acid in the biomarker and a reverse primer to which biotin is bound to the 5'end; Restriction enzymes; Magnetic particles coated with streptavidin; Fluorescent nucleic acid nanostructure; And a biomarker detection kit comprising graphene oxide.
  • the kit may further include tools and/or reagents for collecting biological samples from a subject or patient, as well as tools and/or reagents for preparing genomic DNA, cDNA or RNA from the sample.
  • a labeled oligonucleotide can be used to easily identify it during analysis.
  • the kit may further contain a labeling material such as DNA polymerase and dNTP (dGTP, dCTP, dATP and dTTP), and a fluorescent material.
  • a labeling material such as DNA polymerase and dNTP (dGTP, dCTP, dATP and dTTP)
  • dNTP dGTP, dCTP, dATP and dTTP
  • a pretreatment was performed to make a long-sequence biomarker into a short-length gene biomarker detectable in a three-dimensional fluorescent nanonucleic acid structure.
  • the mutations Del Ex19, L858R, and T790M known as biomarkers specific to lung cancer, were used as examples.
  • the forward primer was designed to amplify a portion adjacent to the target. I did it.
  • a reverse primer a sequence with biotin attached to 5'was designed and then PCR was performed (Table 2).
  • a PCR clean up kit was used to remove the remaining primers and amplification enzymes after amplification.
  • the purified amplification product was reacted with a restriction enzyme corresponding to each mutation (biomarker), and cut into a target portion and a waste portion (Table 1). Thereafter, the reaction was performed with magnetic beads coated with streptavidin for 15 minutes to 4 hours, and the magnetic beads were precipitated using a magnet to obtain a supernatant portion containing the selected targets. Through this, a long-length biomarker was converted into a short-length gene biomarker including its target portion.
  • Example 3 In order to confirm whether the salt concentration condition in the sample pretreatment of Example 1 affects the nucleic acid detection ability of the triangular columnar fluorescent nucleic acid nanostructure of Example 3, a magnetic bead reaction was performed under a salt concentration condition of 1M, and then from 200 mM to A triangular columnar fluorescent nucleic acid nanostructure was prepared at a salt concentration of 1000 mM, and the detection ability of the target nucleic acid obtained by pretreatment with L858R, T790M and Del Ex19 was confirmed.
  • dsDNA of the same length as the target nucleic acid obtained by pretreatment with L858R, T790M and Del Ex19 was used as a control.
  • the triangular column type consisting of a single strand.
  • S1' comprising a sequence complementary to the target sequence c1 (a target sequence pretreated with L858R, T790M or Del Ex19 including the mutation), c2 (GAPDH) and c3 (a sequence pretreated with wild-type L858R, T790M or Del Ex19).
  • S2' and S3' sequences (Table 3) were prepared for each of the pretreated L858R, T790M, and Del Ex19, and the absorbance was measured at a wavelength of 260 nm and quantified.
  • Example 1 The same number of moles (10 pmol) was added so that the molar ratio of all the quantified sequences was 1:1, and the samples pretreated in Example 1 were respectively added. After adjusting the volume so that it is 30 ul per batch, put it in the polymerase chain reaction (PCR) equipment, raise the temperature to 95°C and hold for 5 minutes, lower the temperature to 85°C and hold for 5 minutes, then 1 minute The temperature was lowered by 0.5°C to 20°C and maintained for 5 minutes, and a heat treatment to lower the temperature to 4°C was performed at a salt concentration of 200 mM to 1000 mM.
  • PCR polymerase chain reaction
  • the prepared triangular columnar fluorescent nanonucleic acid structure was put into a fluorescent device (plate reader), and the excitation/emission corresponding to FAM, Cy3 and Cy5 was measured at 485nm/525nm, 540nm/570nm and 640nm/670nm wavelengths, respectively.
  • the ratio of the mutant/wild type of the target biomarker was measured through the measured Cy5/Cy3 fluorescence ratio.
  • the salt concentration of 200 mM to 1000 mM had no effect on the fluorescence value analysis (FIGS. 3 and 4). Since all pretreated samples have a salt concentration of 1000 mM, the more the pretreated sample is used, the higher the salt concentration of the solution when reacting the triangular columnar fluorescent nucleic acid nanostructure with the pretreated sample. When reacting in a solution lower than 1000 mM concentration, the amount of sample that can be treated is limited, and the more samples are used, the greater the fluorescence value can be obtained, so it is advantageous to synthesize the sample and the fluorescent nucleic acid nanostructure at 1000 mM concentration. It was established with the most optimal conditions.
  • Example 3-1 it is specific for the target nucleic acid obtained by pretreatment with L858R, T790M and Del Ex19, but structurally, the bottom surface of the tetrahedron forms a double strand, and the side surface and the portion to be connected to the graphene oxide are single stranded.
  • a tetrahedral fluorescent nanonucleic acid structure (Fig. 5) was synthesized, and in a polymerase chain reaction (PCR) equipment, the temperature was raised to 95°C and maintained for 2 minutes, and the temperature was lowered to 60°C, and then 1°C per minute. The temperature was lowered to 20° C., maintained at 20° C. for 5 minutes, and then produced by performing heat treatment to lower the temperature to 4° C.
  • PCR polymerase chain reaction
  • the tetrahedral fluorescent nucleic acid nanostructure has a sequence U1 (consisting of sequences complementary to each of S1, S2 and S3) on the upper surface (bottom of the tetrahedron) of the structure that will form a total of 4 single-stranded double-stranded structures; And S1, S2 and S3 comprising a sequence complementary to each of the target sequences c1, c2 and c3, and the prepared tetrahedral fluorescent nucleic acid nanostructure-graphene oxide complex is a target on the side in the structure where the bottom of the tetrahedron is raised.
  • a single-stranded portion complementary to and the adenine sequence (poly A) of the S1 strand is elongated and attached to graphene oxide.
  • the detection limit values of Del Ex19, L858R, and T790M which are three target biomarkers, were measured at an optimized salt concentration of 1000 mM.
  • the target concentration was adjusted to 5 to 200 nM to measure the fluorescence intensity by concentration, and the detection limit was calculated according to the guideline EP17 released by Clinical and Laboratory Standards Institute (CLSI).
  • CLSI Clinical and Laboratory Standards Institute
  • the Cy5/Cy3 fluorescence value at this time was confirmed and normalized.
  • the ratio of the Cy5/Cy3 fluorescence values according to the target concentration was analyzed.
  • the concentration of the pretreated total target was fixed at 200 nM, and the ratio of the target with the mutation was changed from 0 to 100%, while Cy3 fluorescence. Values and Cy5 fluorescence values were normalized. In addition, the measured Cy5/Cy3 fluorescence value was used to calculate the minimum identifiable mutation ratio in the target of 200 nM.
  • the Cy5/Cy3 fluorescence ratio was observed as the length of the target sequence including the Del Ex19 mutation was adjusted to 22 to 150 nt (bp). .
  • the triangular columnar fluorescent nucleic acid nanostructure-graphene oxide complex of the present invention is designed to show a low Cy5/Cy3 fluorescence value when wild type is added, and a high Cy5/Cy3 fluorescence value when the mutant type is added.
  • bp 75 to 100 nt
  • a sharp decrease was observed in both the wild type and the mutant. It is inferred that the longer the length, the more unreacted parts except the target site inhibit the reaction between the target sequence and the triangular columnar fluorescent nucleic acid nanostructure.
  • RNA that was not pretreated was used as a control.
  • PCR of RNA extracted from the four cell lines, restriction enzyme treatment (RE), and target sequence purification using streptavidin-biotin binding were confirmed by electrophoresis using 12% polyacrylamide.
  • the pretreatment method of the present invention it is possible to selectively obtain only the mutant portion of the required gene biomarker, thereby minimizing non-specific binding between the target nucleic acid and the fluorescent nucleic acid nanostructure-graphene structure, thereby improving the detection function.
  • Low concentration of biomarker can be effectively diagnosed, and further, there is an effect of early diagnosis of disease from liquid biopsy and easily confirming the effect of drugs.

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Abstract

La présente invention concerne un procédé pour améliorer la détection de multiples biomarqueurs à l'aide d'un complexe d'oxyde en graphène et à nanostructure d'acide nucléique fluorescent par l'intermédiaire d'un procédé de prétraitement d'acide nucléique. Selon un procédé de prétraitement de la présente invention, seule une partie de mutation d'un biomarqueur génétique nécessaire peut être obtenue de manière sélective, et, par conséquent, une liaison non spécifique entre un acide nucléique cible et une structure en graphène à nanostructure d'acide nucléique fluorescent peut être réduite au minimum, ce qui permet d'obtenir une fonction de détection améliorée, et une faible concentration de biomarqueurs peut également être efficacement diagnostiquée. En outre, il y a l'effet de diagnostiquer des maladies précoces par biopsie liquide et de confirmer facilement les effets de médicaments et analogues.
PCT/KR2020/013235 2019-10-15 2020-09-28 Procédé de détection de multiples biomarqueurs Ceased WO2021075764A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20030041415A (ko) * 2001-11-20 2003-05-27 주식회사 바이오메드랩 마이코박테리움 종 구별 및 약제내성 탐지를 위한진단키트 및 그 키트의 제조방법
KR20040105744A (ko) * 2002-03-01 2004-12-16 라브겐, 인코퍼레이티드 게놈 변이의 신속한 분석법
US20090117549A1 (en) * 2006-07-18 2009-05-07 Weihong Tan Aptamer-based methods for identifying cellular biomarkers
KR20120007002A (ko) * 2009-03-15 2012-01-19 리보메드 바이오테크놀로지스, 인코퍼레이티드 압스크립션 기반 분자 검출
KR20190086259A (ko) * 2018-01-12 2019-07-22 성균관대학교산학협력단 핵산 검출용 형광핵산나노구조체-그래핀 바이오센서

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20030041415A (ko) * 2001-11-20 2003-05-27 주식회사 바이오메드랩 마이코박테리움 종 구별 및 약제내성 탐지를 위한진단키트 및 그 키트의 제조방법
KR20040105744A (ko) * 2002-03-01 2004-12-16 라브겐, 인코퍼레이티드 게놈 변이의 신속한 분석법
US20090117549A1 (en) * 2006-07-18 2009-05-07 Weihong Tan Aptamer-based methods for identifying cellular biomarkers
KR20120007002A (ko) * 2009-03-15 2012-01-19 리보메드 바이오테크놀로지스, 인코퍼레이티드 압스크립션 기반 분자 검출
KR20190086259A (ko) * 2018-01-12 2019-07-22 성균관대학교산학협력단 핵산 검출용 형광핵산나노구조체-그래핀 바이오센서

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