WO2022225114A1 - Biocapteur comprenant un complexe nanoparticule d'or-adn et un complexe crispr/cas12a, et procédé de détection d'adn cible à l'aide de celui-ci - Google Patents

Biocapteur comprenant un complexe nanoparticule d'or-adn et un complexe crispr/cas12a, et procédé de détection d'adn cible à l'aide de celui-ci Download PDF

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WO2022225114A1
WO2022225114A1 PCT/KR2021/015153 KR2021015153W WO2022225114A1 WO 2022225114 A1 WO2022225114 A1 WO 2022225114A1 KR 2021015153 W KR2021015153 W KR 2021015153W WO 2022225114 A1 WO2022225114 A1 WO 2022225114A1
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dna
stranded dna
gold
crispr
immobilized
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Korean (ko)
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백세환
최정우
최진하
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솔바이오 주식회사
서강대학교산학협력단
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • C12Q2563/107—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
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/137—Metal/ion, e.g. metal label
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q2563/155—Particles of a defined size, e.g. nanoparticles

Definitions

  • the present invention was made by the project number 1038198800 and project number 10381988 under the support of the Ministry of SMEs and Startups.
  • the title is "Early cancer disease diagnosis kit based on tumor-related exosome target biomarkers in the blood", the lead institution is Sol Bio, and the study period is from 2020-11-01 ⁇ 2021-05-31.
  • the present invention was made by the project specific number 1425149332 and project number S3012455 under the support of the Ministry of SMEs and Startups.
  • the project name is "Early cancer diagnosis based on exosome subgroup biomarker profiling", the lead institution is Sol Bio, and the research period is from 2020-10-01 ⁇ 2022-09-30.
  • the present invention relates to a biosensor including a gold nanoparticle-DNA complex and a CRISPR/Cas12a complex, and a target DNA detection method using the same, and more particularly, to two gold nanoparticles containing single-stranded DNA containing a fluorescent material and
  • a biosensor prepared by immobilizing single-stranded DNA comprising a nucleotide sequence complementary to this, when the target DNA binds to the CRISPR/Cas12a complex, it uses the property of decomposing the surrounding single-stranded DNA to emit fluorescence. It relates to a method capable of detecting target DNA by inducing it.
  • Cas12a (Cpf-1), one of the CRISPR proteins, has the property of randomly decomposing a single DNA strand around it while cutting both ends of the DNA when the target DNA binds. Using this property, a quencher-fluorophore linked to single-stranded DNA is injected around Cas12a that reacts with the target DNA, and DNA degradation and fluorescence emission are induced according to the concentration of the target DNA to induce DNA degradation.
  • a DNA biosensor that measures
  • Such a biosensor using CRISPR/Cas12a has the advantage of being able to measure target DNA up to the aM (attomole) level and measure only the desired DNA due to its excellent sensitivity.
  • aM attomole
  • PCR polymerase chain reaction
  • Fluorescent materials are used as one of the important signal materials in biosensors.
  • the use of a fluorescent material has an advantage in that the presence or absence of a protein can be visually checked.
  • the fluorescent material does not exhibit a fluorescent quenching effect when the distance to the surface of the noble metal nanomaterial is close, so it can be applied to biosensor production by grafting it to the protein or DNA binding phenomenon.
  • the present inventors developed a high-sensitivity DNA biosensor in which a gold nanoparticle-DNA complex production based on a noble metal-based fluorescence amplification effect and CRISPR gene scissors technology are grafted.
  • an object of the present invention is to provide a first gold nanoparticle to which a first single-stranded DNA including a fluorescent material is immobilized at one end, and a base complementary to the first single-stranded DNA immobilized on the first gold nanoparticle.
  • An object of the present invention is to provide a gold nanoparticle-DNA complex including a second gold nanoparticle to which a second single-stranded DNA comprising a sequence is immobilized.
  • Another object of the present invention is to provide a first gold nanoparticle to which a first single-stranded DNA including a fluorescent material is immobilized at one end, and a nucleotide sequence complementary to binding to a first single-stranded DNA immobilized on the first gold nanoparticle.
  • a biosensor comprising a CRISPR/Cas12a complex will provide
  • Another object of the present invention is to provide a method for detecting a target DNA in a sample comprising the steps of:
  • a CRISPR/Cas12a complex comprising a CRISPR/Cas12a (Cpf-1) protein and a guide RNA comprising a region binding to the CRISPR/Cas12a protein and a guide sequence that hybridizes with a target DNA.
  • Another object of the present invention is to provide a first gold nanoparticle to which a first single-stranded DNA including a fluorescent material is immobilized at one end;
  • a CRISPR/Cas12a complex comprising a CRISPR/Cas12a protein and a guide RNA comprising a region binding to the CRISPR/Cas12a protein and a guide sequence that hybridizes with a target DNA.
  • It relates to a target DNA detection use of a biosensor comprising a.
  • the present invention relates to a biosensor comprising a gold nanoparticle-DNA complex and a CRISPR/Cas12a(Cpf-1) complex, and a method for detecting a target DNA using the same.
  • a phenomenon-inducing gold nanoparticle-DNA complex it enables sensitive and selective measurement of DNA, and has excellent fluorescence signal enhancement and sensitivity and selectivity.
  • the present inventors took advantage of the property that CRISPR/Cas12a activated by target DNA degrades random single-stranded DNA so that when single-stranded DNA is degraded, the distance between the gold nanoparticles and the fluorescent material is about 7 nm.
  • a DNA complex was prepared, and the effect of increasing the measurement sensitivity compared to the existing CRISPR gene scissors-based fluorescence quenching-amplification-based target DNA measurement technology was confirmed.
  • a first gold nanoparticle to which a first single-stranded DNA including a fluorescent material is immobilized at one end, and a nucleotide sequence complementary to the first single-stranded DNA immobilized on the first gold nanoparticle A gold nanoparticle-DNA complex including a second gold nanoparticle to which a second single-stranded DNA comprising
  • the first single-stranded DNA may have a length of 7 to 8 nm, and may be composed of 15 to 25 nucleotide sequences, for example, may be composed of 18 to 22 nucleotide sequences. It is not limited. When the length of the first single-stranded DNA exceeds 8 nm, the intensity of fluorescence may be sharply decreased.
  • the second single-stranded DNA may additionally include a portion having a length of 2 to 3 nm that does not complementarily bind to the first single-stranded DNA, and the second single-stranded DNA may consist of 25 to 35 nucleotide sequences. and, for example, may consist of 28 to 32 nucleotide sequences, but is not limited thereto.
  • the first single-stranded DNA may be immobilized on the first gold nanoparticles by a thiol-gold bond.
  • the first single-stranded DNA may be immobilized on the first gold nanoparticles by applying a thiol group to one end other than the one end containing the fluorescent material.
  • the second single-stranded DNA may be immobilized on the second gold nanoparticles by a thiol-gold bond.
  • the diameter of the first gold nanoparticles may be 10 to 25 nm, for example, 15 to 22 nm, but is not limited thereto.
  • the diameter of the first gold nanoparticles is less than 10 nm, the fluorescence amplification effect expressed from the gold nanoparticle-DNA complex may not be sufficient.
  • the diameter of the second gold nanoparticles may be 50 to 70 nm, for example, 55 to 65 nm, but is not limited thereto.
  • the first gold nanoparticles When the first gold nanoparticles have a relatively smaller diameter than the second gold nanoparticles, they exist in a larger number in actual application. More single-stranded DNA can be added, which can increase the efficiency of the process.
  • Another aspect of the present invention is a first gold nanoparticle to which a first single-stranded DNA including a fluorescent material is immobilized at one end,
  • a second gold nanoparticle immobilized with a second single-stranded DNA comprising a nucleotide sequence complementary to the first single-stranded DNA immobilized on the first gold nanoparticle
  • a CRISPR/Cas12a complex comprising a CRISPR/Cas12a protein and a guide RNA comprising a region binding to the CRISPR/Cas12a protein and a guide sequence that hybridizes with a target DNA.
  • a biosensor comprising
  • the first single-stranded DNA may have a length of 7 to 8 nm
  • the second single-stranded DNA additionally includes a portion having a length of 2 to 3 nm that does not complementarily bind to the first single-stranded DNA.
  • the first single-stranded DNA may be immobilized on the first gold nanoparticles by a thiol-gold bond.
  • the first single-stranded DNA may be immobilized on the first gold nanoparticles by applying a thiol group to one end other than the one end containing the fluorescent material.
  • the second single-stranded DNA may be immobilized on the second gold nanoparticles by a thiol-gold bond, and may not hybridize with the guide RNA sequence.
  • the diameter of the first gold nanoparticles may be 10 to 25 nm, for example, 15 to 22 nm, but is not limited thereto.
  • the diameter of the second gold nanoparticles may be 50 to 70 nm, for example, 55 to 65 nm, but is not limited thereto.
  • the distance between the gold nanoparticles and the fluorescent material is about 7 nm.
  • a gold nanoparticle-DNA complex including the first gold nanoparticles and the second gold nanoparticles was prepared.
  • a fluorescent material is attached to one end of the first single-stranded DNA immobilized on the relatively small first gold nanoparticles having a size of 20 nm, and the second single-stranded DNA is complementary to the first single-stranded DNA. is immobilized on a relatively large 60 nm-sized second gold nanoparticle, and the two gold nanoparticles are connected to each other by complementary binding of two single-stranded DNAs immobilized thereon.
  • the pair of DNAs have different sizes, and the lengths of the double-stranded DNA part and the single-stranded DNA part based on the complementary binding form are about 7 nm and 2 nm, respectively, so that between the fluorescent material and the second gold nanoparticles The distance of is maintained at 2-3 nm equal to the length of the single-stranded DNA portion. Accordingly, the fluorescence material present therebetween was designed to be positioned close to the second gold nanoparticles at a distance of 2 nm to quench the fluorescence.
  • Another aspect of the present invention is a method for detecting a target DNA in a sample comprising the steps of:
  • a CRISPR/Cas12a complex comprising a CRISPR/Cas12a protein and a guide RNA comprising a region binding to the CRISPR/Cas12a protein and a guide sequence that hybridizes with a target DNA.
  • the first single-stranded DNA may have a length of 7 to 8 nm
  • the second single-stranded DNA additionally includes a portion having a length of 2 to 3 nm that does not complementarily bind to the first single-stranded DNA.
  • the first single-stranded DNA may be immobilized on the first gold nanoparticles by a thiol-gold bond.
  • the first single-stranded DNA may be immobilized on the first gold nanoparticles by applying a thiol group to one end other than the one end containing the fluorescent material.
  • the second single-stranded DNA may be immobilized on the second gold nanoparticles by a thiol-gold bond, and may not hybridize with the guide RNA sequence.
  • the diameter of the first gold nanoparticles may be 10 to 25 nm, for example, 15 to 22 nm, but is not limited thereto.
  • the diameter of the second gold nanoparticles may be 50 to 70 nm, for example, 55 to 65 nm, but is not limited thereto.
  • the fluorescent material is the second gold
  • the state was switched from the quenched state to the amplified state.
  • CRISPR/Cas12a can degrade single-stranded DNA when CRISPR RNA and target DNA are complementarily bound, it can be used to perform selective measurement of target DNA.
  • the present invention relates to a biosensor comprising a gold nanoparticle-DNA complex and a CRISPR/Cas12a complex, and a method for detecting a target DNA using the same, wherein the biosensor is a gold nanoparticle-inducing extinction-amplification phenomenon of a fluorescence emission signal- As a DNA complex, it is possible to measure DNA sensitively and selectively, improving fluorescence signal, and has excellent sensitivity and selectivity. By effectively applying it to the detection of target DNA, cell-based non-destructive testing, real-time measurement, and body fluid-based extended disease diagnosis kit etc. can be used.
  • FIG. 1 is a schematic diagram of a gold nanoparticle-DNA complex prepared according to an embodiment of the present invention.
  • TEM 2 is a transmission electron microscope (TEM) of 20 nm and 60 nm gold nanoparticles (top) and 20 nm and 60 nm gold nanoparticles (bottom) on which DNA is immobilized according to an embodiment of the present invention; ) is an image.
  • TEM transmission electron microscope
  • FIG. 3 is a schematic diagram showing the effect of metal-enhanced fluorescence (MEF) due to CRISPR/Cas12a implemented in the gold nanoparticle-DNA complex prepared according to an embodiment of the present invention.
  • MEF metal-enhanced fluorescence
  • ssDNA single-stranded DNA
  • FITC Fluorescein isothiocyanate
  • Au-FITC gold nanoparticles
  • It is a graph measuring the fluorescence intensity when single-stranded DNA is immobilized on nanoparticles (60 Au), when two nanoparticles are combined (Before), and when the reaction proceeds due to CRISPR/Cas12a (After).
  • FIG. 5 is a graph showing fluorescence intensity by confirming the reaction results for each concentration of BRCA-1, a target DNA, according to an embodiment of the present invention.
  • SM single-mismatched DNA
  • DM double-mismatched DNA
  • microRNA-21 microRNA-21
  • the present invention provides a first gold nanoparticle to which a first single-stranded DNA containing a fluorescent material is immobilized at one end, and a nucleotide sequence complementary to the first single-stranded DNA immobilized on the first gold nanoparticle.
  • the present invention relates to a gold nanoparticle-DNA complex comprising a second gold nanoparticle to which a second single-stranded DNA is immobilized.
  • % used to indicate the concentration of a specific substance is (weight/weight)%, solid/liquid (weight/volume)%, and (weight/volume)%, and Liquid/liquid is (vol/vol)%.
  • CRISPR/Cas12a has the property of decomposing the surrounding single-stranded DNA (ssDNA) when the target DNA binds complementary to the CRISPR RNA.
  • ssDNA single-stranded DNA
  • a gold nanoparticle-DNA complex that can be used as a sensing probe was prepared.
  • the gold nanoparticles used in this study were performed by pairing gold nanoparticles with a diameter of 20 nm and 60 nm, respectively, sold by BBInternational.
  • a gold nanoparticle-DNA nanocomposite was prepared by immobilizing single-stranded DNA capable of complementary binding to the gold nanoparticle.
  • Each single-stranded DNA was attached to the surface of gold nanoparticles using a specific thiol-gold reaction, and gold nanoparticles of different sizes were attached to each other using complementary DNA binding.
  • the fluorescent material was attached to the tip of single-stranded DNA immobilized on gold nanoparticles with a size of 20 nm.
  • the sequences of single-stranded DNA used for immobilization are shown in Table 1 below.
  • the gold nanocomposite separated through the same washing process as the 20 nm gold nanoparticles was then passivated (4°C overnight) using a 3% bovine serum albumin (BSA) solution. This is an essential process to prevent aggregation of gold nanoparticles and increase the selectivity of the sensor.
  • BSA bovine serum albumin
  • TEM transmission electron microscope
  • Gold nanoparticles have the property of amplifying the fluorescence emission intensity of a fluorescent material due to the surface plasmon resonance effect. These characteristics are expressed only when a specific distance between the gold nanoparticles and the fluorescent material is secured.
  • the fabricated gold nanoparticle-DNA complex is a biosensor system designed to dramatically increase fluorescence intensity through fluorescence quenching and metal-enhanced fluorescence (MEF) effects. More specifically, when the fluorescent material maintains a distance of several nanometers or less from the surface of a noble metal such as gold, a quenching effect in which the fluorescent signal is lost occurs. On the other hand, as shown in FIG. 3, if the absorption wavelength of the gold nanoparticles and the emission wavelength of the fluorescent material coincide with maintaining a constant distance of about 7-8 nm, the emission intensity of the fluorescent material can be amplified by several orders of magnitude. have. This is called the fluorescence amplification effect.
  • the biomaterial that binds the gold nanoparticles and the fluorescent material is DNA immobilized on both gold nanoparticles.
  • the two complementary DNAs are bonded to each other, part double-stranded DNA part and the other part single-stranded DNA part. It is located close to the surface of 60 nm gold nanoparticles (about 2 nm) and exhibits fluorescence quenching.
  • Example 3 Invented gold nanoparticle-DNA complex-based target DNA quantification
  • the prepared gold nanoparticle-DNA complex showed a phenomenon in which the fluorescence intensity was changed depending on whether the single-stranded DNA part was decomposed. Based on these results, in the present invention, an attempt was made to quantify the target DNA using CRISPR/Cas12a, which selectively binds to the target DNA and exhibits the degradation characteristics of the single-stranded DNA portion.
  • the target DNA that can be measured in the gold nanoparticle-DNA complex can be measured as long as it is DNA composed of approximately 20 base sequences, and BRCA-1, one of representative cell-free DNAs, was used as an example.
  • This DNA is DNA released in mutant form from breast cancer cells, and BRCA-1 having a normal sequence can be measured in normal cells, so it can be used as a biomarker for breast cancer.
  • the fluorescence intensity was measured to compare the fluorescence intensity for each concentration. More specifically, from 1 fM to 1 nM (1 fM, 10 fM, 100 fM, 1,000 fM, 10,000 fM, 100,000 fM, 1,000,000 fM) of BRCA-1 DNA was tested as a reference, gold nanoparticles-DNA complexes After reacting with and at room temperature for 30 minutes, the fluorescence intensity of 520 nm was measured using a fluorescence measuring instrument.
  • the fluorescence spectrum is a graph showing the fluorescence intensity after CRISPR/Cas12a reaction with BRCA-1 DNA, and it was confirmed that the fluorescence intensity increased linearly as the concentration increased. A linear relationship between the signals was also confirmed. It was confirmed that the measurable DNA concentration was from about 1 fM to 100 pM (10,000 fM) when compared with the control value, and when it exceeded 100 pM, it was confirmed that the fluorescence signal was saturated. .
  • Example 4 Invented gold nanoparticle-DNA complex-based off-target DNA selectivity test
  • RNA-141 single-sequence mismatched DNA
  • DM double-mismatched DNA
  • miR-21 microRNA-21
  • Target target DNA
  • the gold nanoparticle-based fluorescence amplification biosensor can selectively measure target DNA using the characteristics of CRISPR/Cas12a.
  • the present invention relates to a biosensor including a gold nanoparticle-DNA complex and a CRISPR/Cas12a complex, and a target DNA detection method using the same, and more particularly, to two gold nanoparticles containing single-stranded DNA containing a fluorescent material and
  • a biosensor prepared by immobilizing single-stranded DNA comprising a nucleotide sequence complementary to this, when the target DNA binds to the CRISPR/Cas12a complex, it uses the property of decomposing the surrounding single-stranded DNA to emit fluorescence. It relates to a method capable of detecting target DNA by inducing it.

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Abstract

La présente invention concerne un biocapteur comprenant un complexe nanoparticule d'or-ADN et un complexe CRISPR/Cas12a, et un procédé de détection d'ADN cible à l'aide de celui-ci. Le biocapteur comprend un complexe nanoparticule d'or-ADN induisant l'extinction-amplification d'un signal d'émission de fluorescence et permettant la mesure sensible et sélective de l'ADN, présentant ainsi un signal de fluorescence amélioré et ayant une excellente sensibilité et sélectivité. Par conséquent, le biocapteur peut être appliqué efficacement à la détection de l'ADN cible, pouvant ainsi être utilisé comme kits pour les tests non destructifs basés sur les cellules, la mesure en temps réel et le diagnostic étendu des maladies basé sur les fluides corporels, et analogues.
PCT/KR2021/015153 2021-04-23 2021-10-27 Biocapteur comprenant un complexe nanoparticule d'or-adn et un complexe crispr/cas12a, et procédé de détection d'adn cible à l'aide de celui-ci WO2022225114A1 (fr)

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KR1020210053086A KR102512999B1 (ko) 2021-04-23 2021-04-23 금 나노 입자-DNA 복합체 및 CRISPR/Cas12a 복합체를 포함하는 바이오센서, 및 이를 이용한 표적 DNA 검출 방법

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