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WO2018151138A1 - Sonde à acide nucléique pour détecter une substance cible - Google Patents

Sonde à acide nucléique pour détecter une substance cible Download PDF

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Publication number
WO2018151138A1
WO2018151138A1 PCT/JP2018/005032 JP2018005032W WO2018151138A1 WO 2018151138 A1 WO2018151138 A1 WO 2018151138A1 JP 2018005032 W JP2018005032 W JP 2018005032W WO 2018151138 A1 WO2018151138 A1 WO 2018151138A1
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nucleic acid
stranded nucleic
region
acid probe
target substance
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PCT/JP2018/005032
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English (en)
Japanese (ja)
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章 和田
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国立研究開発法人理化学研究所
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Priority to JP2018568555A priority Critical patent/JP7176739B2/ja
Publication of WO2018151138A1 publication Critical patent/WO2018151138A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • 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
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present invention relates to a nucleic acid probe for detecting a target substance, and a method for determining the presence or absence of a target substance or measuring the amount using the probe.
  • Enzyme-linked immunosorbent assays using antibodies are widely used, ranging from diagnosis of viruses, protozoa, and other infections, allergies, and autoimmune diseases to detection of various cancer biomarkers. ing.
  • ELISA Enzyme-linked immunosorbent assays
  • Non-Patent Document 1 Aptamer is a general term for single-stranded DNA, RNA, peptides, and the like that exhibit high affinity and specificity for any target molecule. Aptamer target binding is derived from the complex and tiny three-dimensional structure formed by aptamers, and it has been reported that it can bind to various target molecules such as proteins, peptides, low molecular weight compounds, and metal ions. Yes.
  • aptamers developed as practical biosensing elements.
  • One of the reasons is that the introduction of aptamers such as labels and radioisotopes used to detect aptamers is not simple, and the detection sensitivity and / or specificity are not necessarily sufficient. Met.
  • An object of the present invention is to provide a nucleic acid probe that can be easily prepared and can be detected with high sensitivity. Another object of the present invention is to provide a method for determining the presence of a target substance or measuring the amount using the nucleic acid probe.
  • the present inventor has found that a probe capable of detecting a target substance with high sensitivity can be prepared by adding a double-stranded part into which an intercalator can be inserted into a target binding region, and has completed the present invention.
  • a first single strand comprising a target binding region that specifically binds to a target substance, and a first single-stranded nucleic acid region comprising two or more bases linked to either end of the target binding region
  • a second single-stranded nucleic acid molecule comprising a second single-stranded nucleic acid region comprising two or more bases complementary to the base sequence of the first single-stranded nucleic acid region
  • the first single-stranded nucleic acid region and the second single-stranded nucleic acid region are base-paired to form a detection region into which an intercalator can be inserted.
  • Nucleic acid probe for target substance detection is provided.
  • nucleic acid probe according to (1) wherein the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule are linked at the end.
  • the intercalator is ethidium bromide, psoralen, acridine, acridine orange, proflavine, propidium iodide, 7-aminoactinomycin D, actinomycin D, DAPI, EvaGreen (registered trademark), DRAQ7 TM , SYTOX (registered trademark) ), Midori Green, SYBR (registered trademark) Green I, SYBR (registered trademark) Green II, and SYBR (registered trademark) Gold, the nucleic acid according to any one of (1) to (9) probe.
  • (11) contacting the nucleic acid probe according to any one of (1) to (10) with a sample;
  • a method for detecting the presence or absence of a target substance comprising: adding an intercalator to a sample and inserting the sample into a nucleic acid probe; and detecting the nucleic acid probe based on the intercalator.
  • (12) The method according to (11), wherein the detection step is performed by gel shift assay or flow cytometry.
  • (13) a step of bringing the nucleic acid probe according to any one of (1) to (10) into contact with a sample; Adding an intercalator to the sample and inserting it into the nucleic acid probe; Measure the amount of the target substance, including the step of measuring the amount of the nucleic acid probe bound to the target substance based on the intercalator and the step of estimating the amount of the target substance based on the amount of the nucleic acid probe bound to the target substance.
  • Method. (14) The method according to (13), wherein the measurement step is performed by gel shift assay or flow cytometry.
  • a probe capable of detecting a target substance with high sensitivity is provided, and a simple and highly sensitive method for detecting and / or measuring a target substance using the probe can be provided.
  • FIG. 1 is a conceptual diagram showing an embodiment of the nucleic acid probe of the present invention.
  • 101 indicates a first single-stranded nucleic acid molecule
  • 102 indicates a second single-stranded nucleic acid molecule.
  • Reference numerals 103 and 106 respectively denote first and second single-stranded nucleic acid regions, which form a detection region into which an intercalator can be inserted by base pairing (shown by a solid line).
  • 104 denotes a spacer sequence that can be optionally included, and 105 denotes a target binding region that specifically binds to a target substance.
  • FIG. 1B shows a nucleic acid probe that differs from the nucleic acid probe shown in FIG.
  • FIG. 2 is a conceptual diagram showing an embodiment of the nucleic acid probe of the present invention.
  • the ends of the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule are linked to form a single-stranded nucleic acid probe as a whole.
  • the 5 ′ end of the first single-stranded nucleic acid region 203 and the 3 ′ end of the second single-stranded nucleic acid region 206 are connected via a connecting portion 207.
  • 204 represents a spacer sequence that can be optionally included
  • 205 represents a target binding region that specifically binds to a target substance.
  • FIG. 2B shows a nucleic acid probe that differs from the nucleic acid probe shown in FIG. 2A only in the direction of 5 ′ ⁇ 3 ′.
  • FIG. 2C shows the 5 ′ end of the second single-stranded nucleic acid region 206 and the 3 ′ end of the target binding region 205 are connected via a connecting portion 208.
  • FIG. 2D shows a nucleic acid probe that differs from the nucleic acid probe shown in FIG. 2C only in the 5 ′ ⁇ 3 ′ direction.
  • FIG. 3 is a conceptual diagram showing one embodiment of the nucleic acid probe of the present invention.
  • the end of the first single-stranded nucleic acid region 303 is connected to the end of the second single-stranded nucleic acid region 306 via the connecting portion 307, and the end of the second single-stranded nucleic acid region 306 is connected.
  • the ends of the target binding region 305 are linked via a linking portion 308 to form a circular single-stranded nucleic acid probe as a whole.
  • FIG. 4 shows the results of evaluation by gel shift assay of the binding between the nucleic acid molecule S1mt-dsRNA (200) or S1mt-dsRNA (400) prepared in Example and the target molecule.
  • lane 1 hybridizes S1mt-ssRNA (200) (1.2 pmol)
  • lane 2 contains C-ssRNA (200) (1.2 pmol) containing its complementary sequence
  • lane 3 hybridizes these two nucleic acid molecules.
  • the electrophoresis result of S1mt-dsRNA (200) (0.3 pmol) made soybean is shown.
  • Lane 4 shows the results of a solution containing S1mt-dsRNA (200) (0.3 pmol) and Streptavidin (3 ⁇ pmol) .
  • Lane 5 shows a mixture of S1mt-dsRNA (200) (0.3 pmol) and hDFHR (3 pmol).
  • the lanes 6 to 7 show the results of the prepared solutions, and the results of the solutions obtained by mixing A8-dsRNA (200) (0.3 pmol) alone or Streptavidin (3 pmol).
  • lane 1 is S1mt-ssRNA (400) (1.2 pmol)
  • lane 2 is C-ssRNA (400) (1.2 ⁇ ⁇ pmol) containing its complementary sequence
  • lane 3 is these two nucleic acid molecules.
  • the electrophoresis result of S1mt-dsRNA (400) (0.3 pmol) hybridized with is shown.
  • Lane 4 shows the results of a solution containing S1mt-dsRNA (400) (0.3 pmol) and Streptavidin (3 pmol) .
  • Lane 5 shows a mixture of S1mt-dsRNA (400) (0.3 pmol) and hDFHR (3 pmol).
  • the lanes 6 to 7 show the results of the solution obtained by mixing A8-dsRNA (400) (0.3 pmol) alone or a mixture thereof with Streptavidin (3 pmol).
  • the arrowhead indicates a band formed by binding of the nucleic acid molecule and the target protein, streptavidin.
  • M represents a marker (hereinafter the same applies to FIGS. 5 to 6).
  • Lanes 1 and 2 show the results of a solution prepared by mixing A8-dsRNA (200) as a control and translation solution (1.4 ⁇ l) or Bap-GS2-Halo-F (1.4 ⁇ l), respectively.
  • Lanes 3 and 4 show the results of a solution obtained by mixing b-dsRNA (200) and translation solution (1.4 ⁇ l) or Bap-GS2-Halo-F (1.4 ⁇ l), respectively.
  • Lanes 5 and 6 show the results of a solution obtained by mixing bb-dsRNA (200) and translation solution (1.4 ⁇ l) or Bap-GS2-Halo-F (1.4 ⁇ l), respectively.
  • the arrowhead indicates a band formed by binding of the nucleic acid molecule and the target protein Bap-GS2-Halo-F.
  • FIG. 6 shows the results of gel shift assay evaluating the binding between the nucleic acid molecule bb-dsRNA (400) prepared in Example and the target molecule.
  • Lanes 1 and 2 show the results of a solution obtained by mixing bb-dsRNA (400) and translation solution (1.4 ⁇ l) or Bap-GS2-Bap-GS2-Halo-F (1.4 ⁇ l), respectively.
  • Lanes 3 and 4 show the results of a solution obtained by mixing bb-dsRNA (400) and translation solution (2.7 ⁇ l) or Bap-GS2-Bap-GS2-Halo-F (2.7 ⁇ l), respectively.
  • the arrowhead indicates a band formed by binding of the nucleic acid molecule and the target protein Bap-GS2-Bap-GS2-Halo-F.
  • nucleic acid probe for target substance detection relates to a target substance detection nucleic acid probe comprising a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule.
  • the nucleic acid probe of the present invention includes a target binding region in the first single-stranded nucleic acid molecule and includes a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule as described in detail below. Since the single-stranded nucleic acid regions contained in each form a base pair with each other to form a detection region (double-stranded portion), the intercalator can be used to detect the target substance.
  • nucleic acid or “nucleic acid molecule” means a biological macromolecule composed of nucleotides as a principle unit and linked by phosphodiester bonds, and “nucleic acid probe” detects a target molecule. Means a nucleic acid molecule.
  • the “nucleic acid probe” of the present invention may be any of a DNA probe, an RNA probe, and a DNA / RNA mixed probe, but is preferably an RNA probe.
  • the first single-stranded nucleic acid molecule comprises a target binding region that specifically binds to a target substance, and a first single-stranded nucleic acid region comprising two or more bases linked to either end of the target binding region including.
  • the “target binding region” is not limited as long as it is a nucleic acid that specifically binds to a target.
  • An aptamer for example, an RNA aptamer composed of RNA or a DNA aptamer composed of DNA is preferable.
  • the aptamer that can serve as the target binding region may be an existing aptamer whose specific binding property to the target substance is known, or may be a novel aptamer that specifically binds to the target substance.
  • the novel aptamer can be produced by a known method such as SELEX (systematicsystemevolution of ligands by exponential enrichment) method.
  • a nucleic acid molecule bound to a target molecule is selected from a pool consisting of a random sequence region and nucleic acid molecules having primer binding regions at both ends, amplified after recovery, and then transferred to the nucleic acid pool of the next round.
  • This is a method of selecting a nucleic acid having a stronger binding force to a target molecule by repeating a series of cycles of several to several tens of rounds (for example, Tuerk C, Gold L (1990) Science 249 (4968): 505- See 510).
  • the type of “target substance” is not particularly limited, and is a protein (for example, an enzyme, a disease marker such as a cancer marker, and a pathogen toxin), a peptide (for example, a nucleic acid-binding peptide), a low molecular compound, a lipid, a mineral Any of a homo or hetero complex, a metal material and a metal ion may be used.
  • a protein for example, an enzyme, a disease marker such as a cancer marker, and a pathogen toxin
  • a peptide for example, a nucleic acid-binding peptide
  • a low molecular compound for example, a lipid, a mineral Any of a homo or hetero complex, a metal material and a metal ion may be used.
  • the “target binding region” may be a nucleic acid sequence recognized by the nucleic acid binding peptide.
  • nucleic acid binding peptides and nucleic acid sequences recognized by the peptides include Bap ( ⁇ boxB binding peptide) and boxB ( ⁇ phage-derived RNA motif), and transcription factors and binding regions (for example, promoters or enhancers). Etc.
  • the length of the target binding region is not limited and is, for example, 150 bases or less, preferably 100 bases or less, 90 bases or less, 80 bases or less, 70 bases or less, 60 bases or less, or 50 bases or less.
  • the first single-stranded nucleic acid molecule may contain only one target binding region, or 2 or more, for example 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2 May be included. Inclusion of two or more target binding regions in the first single-stranded nucleic acid molecule can increase the detection sensitivity and / or specificity of the nucleic acid probe. Moreover, a probe capable of detecting two or more substances can be prepared by combining different target binding regions. When two or more target binding regions are included, each target binding region may be directly linked, or may be linked via a spacer sequence described later.
  • the first single-stranded nucleic acid region may be directly linked to one end of the target binding region or may be linked via a spacer sequence.
  • the spacer sequence By including the spacer sequence, the degree of freedom of each region is increased, and the influence that the first single-stranded nucleic acid region can have on the three-dimensional structure of the target binding region can be reduced or eliminated.
  • the sequence of the spacer is not limited, but may include, for example, 2 to 20, 4 to 12, 6 to 10, 7 to 9, or 8 adenines.
  • the second single-stranded nucleic acid molecule comprises two or more bases complementary to the base sequence of the first single-stranded nucleic acid region, and the first single-stranded nucleic acid region and the second single-stranded nucleic acid region As a result of base pairing, a detection region (double-stranded portion) into which an intercalator can be inserted is formed.
  • Examples of the first single-stranded nucleic acid molecule contained in the nucleic acid probe of the present invention include, but are not limited to, S1mt-ssRNA (200) (SEQ ID NO: 12), S1mt-ssRNA (400) (SEQ ID NO: 14) a nucleic acid molecule comprising the base sequence of b-ssRNA (200) (SEQ ID NO: 18), bb-ssRNA (200) (SEQ ID NO: 19), or bb-ssRNA (400) (SEQ ID NO: 20) Can be mentioned.
  • Examples of the second single-stranded nucleic acid molecule contained in the nucleic acid probe of the present invention include, but are not limited to, C-ssRNA (200) (SEQ ID NO: 13) or C-ssRNA (400) (sequence) And a nucleic acid molecule comprising the base sequence of No. 15).
  • the number of base pairs in the first and second single-stranded nucleic acid regions and the detection region generated by base pairing of these regions is not particularly limited as long as the number of base pairs in the intercalator is 2 or more.
  • the longer one is preferable for increasing the detection sensitivity, and the shorter one is preferable for reducing the preparation cost. Therefore, it can be appropriately determined in consideration of these factors.
  • the number of base pairs in the first and second single-stranded nucleic acid regions or the number of base pairs in the detection region is preferably 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 30 or more 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, or 200 or more, 5000 or less, 4000 or less, 3000 or less, 2000 or less, 1000 or less, 900 or less, 800 or less 700 or less, 600 or less, 500 or less, 450 or less, or 400 or less, for example, 2 to 5000, 10 to 4000, 20 to 2000, 50 to 1000, 100 to 500, or 200 to 400.
  • the detection region has a plurality of structures such as mismatches, bulges, and internal loops, for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5 Particularly preferably, it may have several, for example 1 to 4, or 1 to 3, or 1 or 2.
  • the nucleic acid probe of the present invention has one or more, for example, 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1 as long as the functions of the target binding region and the detection region are not hindered.
  • -3, 1-2, or 1 modified base and / or artificial base may be included.
  • the modified base and / or the artificial base is preferably included in the target binding region.
  • modified base means a base that has been chemically chemically modified.
  • Modified bases include, for example, modified pyrimidines (eg, 5-hydroxycytosine, 5-fluorouracil, 4-thiouracil, 5- (3-indole-2-ethyl) uracil, 5- (4-hydroxyphenyl-2-ethyl) Uracil), modified purines (eg 6-methyladenine, 6-thioguanosine) and other heterocyclic bases.
  • modified pyrimidines eg, 5-hydroxycytosine, 5-fluorouracil, 4-thiouracil, 5- (3-indole-2-ethyl) uracil, 5- (4-hydroxyphenyl-2-ethyl) Uracil
  • modified purines eg 6-methyladenine, 6-thioguanosine
  • an “artificial base” or “base analog” is an artificially constructed chemical substance having properties similar to those of a natural base constituting a natural nucleotide, and is similar to a natural base.
  • a chemical substance having a base analog (hereinafter referred to as “complementary artificial base”) that can form an artificial base pair.
  • “artificial base pairing” refers to base pairing formed by a pair of complementary artificial bases such as adenine and thymine, adenine and uracil, or guanine and cytosine, which are natural bases.
  • Artificial base pairing is based on chemical bonds via hydrogen bonds found in base pairing between natural bases, physical bonds via fitting based on the molecular structure between artificial bases, and hydrophobic interactions.
  • the artificial base include Ds (7- (2-thienyl) -3H-imidazo [4,5-b] pyridine-3-yl), Pn (2-nitropyrrole-1-yl), Pa (2- formyl-1H-pyrrole-1-yl), P (2-amino-imidazo [1,2-a] -1,3,5-triazin-4 (8H) -one), Z (6-amino-5- nitro-2 (1H) -pyridone), 5SICS (6-methylisoquinoline-1 (2H) -thione), NaM (3-methoxynaphthalen-2-yl), MMO2 (2-methoxy-4-methylphenyl), etc. .
  • the nucleic acid probe of the present invention is modified by adding other substances such as polyethylene glycol (PEG) (for example, PEG polymer of about 20 to about 60 kDa), amino acids, peptides, invertedindT, and lipids. Also good. These substances may be linked via a known linker as necessary. Examples of the linker in the present specification include a nucleotide linker and a peptide linker. It is known that the half-life of a DNA aptamer can generally be extended by linking PEG.
  • PEG polyethylene glycol
  • an “intercalator” is a compound that can bind to a double-stranded nucleic acid and, in a narrow sense, refers to a compound that is inserted between a plane formed by nucleic acid base pairs.
  • intercalators examples include ethidium bromide, psoralen, acridine, acridine orange, proflavine, propidium iodide, 7-aminoactinomycin D, actinomycin D, DAPI, EvaGreen®, DRAQ7 TM , SYTOX (R), Midori Green, SYBR (R) Green I, SYBR (R) Green II, and SYBR (R) Gold.
  • the intercalator may be either cell membrane permeable or cell membrane impermeable.
  • a cell membrane-permeable intercalator By using a cell membrane-permeable intercalator, target substances inside and outside the cell can be detected, while by using a cell membrane-impermeable intercalator, only cells that express the target substance on the cell surface can be detected. Can do.
  • cell membrane impermeable intercalators include DAPI, propidium iodide, acridine orange, EvaGreen (registered trademark), DRAQ7 TM , SYTOX (registered trademark).
  • the direction of 5 ′ ⁇ 3 ′ between the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule is not limited.
  • the first single-stranded nucleic acid molecule is 5 ′ to 3 as shown in FIG. 1A.
  • ′ May include a first single-stranded nucleic acid region 103, an optional spacer sequence 104, and a target binding region 105, or from 3 ′ to 5 ′ as shown in FIG. It may comprise a single stranded nucleic acid region 103, a spacer sequence 104 that may optionally be included, and a target binding region 105.
  • the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule may be linked at the end.
  • the 5 ′ end of the first single-stranded nucleic acid region 203 and the 3 ′ end of the second single-stranded nucleic acid region 206 are connected via a connecting portion 207.
  • the connecting portion 207 is illustrated for convenience, but the 5 ′ end of the first single-stranded nucleic acid region 203 and the 3 ′ end of the second single-stranded nucleic acid region 206 have base sequences. You may connect directly, without interposing.
  • FIG. 2B is a nucleic acid probe that differs from FIG. 2A only in the 5 ′ ⁇ 3 ′ direction.
  • FIG. 2C the 5 ′ end of the second single-stranded nucleic acid region 206 and the 3 ′ end of the target binding region 205 are connected via the connecting portion 208.
  • the linking part 208 is illustrated for convenience, but the 5 ′ end of the second single-stranded nucleic acid region 206 and the 3 ′ end of the target binding region 205 are directly linked without using a base sequence. May be. However, in order to increase the degree of freedom of each region, an embodiment in which the connecting portion 208 is present is more preferable, and the connecting portion 208 has, for example, 2 to 20, 4 to 12, 6 to 10, 7 to 9, or 8 bases. A sequence, such as the spacer sequence described above.
  • FIG. 2D shows a nucleic acid probe that differs from the nucleic acid probe shown in FIG. 2C only in the 5 ′ ⁇ 3 ′ direction.
  • both ends of the first single-stranded nucleic acid molecule may be linked to both ends of the second single-stranded nucleic acid molecule to form a circular single-stranded nucleic acid probe.
  • the 5 ′ ⁇ 3 ′ direction of each single-stranded nucleic acid molecule is not limited.
  • the nucleic acid probe of the present invention can be prepared by methods known in the art such as chemical synthesis.
  • the chemical synthesis method can be performed according to a solid phase synthesis method. Specifically, for example, the chemical synthesis method described in Current Protocols in Nucleic Acid Chemistry, Volume 1, Section 3, Verma S. and Eckstein F., 1998, Annul Rev. Biochem., 67, 99-134 is used. do it.
  • many life science manufacturers for example, Takara Bio, Fasmac, Life Technology, Gene Design, Sigma Aldrich, etc. contract manufacturing They provide services and can use them.
  • RNA can also be prepared by transcription from a template DNA such as a plasmid.
  • the nucleic acid probe can also be prepared by transcription from a template DNA such as a plasmid.
  • two single-stranded nucleic acid region portions can be prepared by using a part of each plasmid-derived double-stranded sequence as a template. In this case, the sequence of the single-stranded nucleic acid region that forms the detection region is derived from the sequence of the plasmid.
  • first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule When linking the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule, it may be prepared as a linked single-stranded molecule, or after each molecule is prepared, it is ligated with ligase or the like. May be.
  • the prepared single-stranded nucleic acid molecule is preferably purified by a method known in the art before use.
  • the purification method include gel purification method, affinity column purification method, HPLC method and the like.
  • the prepared single-stranded nucleic acid molecule forms a detection region (double-stranded part) by a hybridization process.
  • Hybridization can be performed by a known method. For example, first and second single-stranded nucleic acid molecules are incubated in a buffer such as MOPS buffer at a high temperature such as 90 to 95 ° C. for several minutes, and then at room temperature. This can be done by leaving it to stand for more than 30 minutes and slowly cooling it.
  • the nucleic acid probe of the present invention forms a detection region into which an intercalator can be inserted, a target substance can be easily detected using an intercalator signal, for example, fluorescence.
  • an intercalator signal for example, fluorescence.
  • false positives can be reduced, so that highly accurate detection is possible.
  • the detection sensitivity and / or specificity can be further increased by lengthening the detection region as necessary.
  • the probe of the present invention may have one or more of the following advantages (1) to (3): (1)
  • the double-stranded portion contained in the detection region has a degree of freedom. Unlike a single-stranded part with a high level, it has a stable space in which fluorescent molecules remain bound, so that reproducibility and high sensitivity can be detected by intercalation.
  • Double-stranded part is uniform Since a stable structure is maintained, steric hindrance to the target binding region is unlikely to occur.
  • Probes including a double-stranded part having a uniform structure are used in electrophoresis using gels, capillaries, etc. Since it can be separated at a specific position with good reproducibility, it can be detected and quantified specifically and easily.
  • the present invention relates to a method for detecting the presence or absence of a target substance using the nucleic acid probe described in “1. Target substance detection nucleic acid probe”.
  • the step of bringing the nucleic acid probe into contact with the sample contacting step
  • the step of adding an intercalator to the sample and inserting it into the nucleic acid probe insertion step
  • the step of detecting the nucleic acid probe based on the intercalator detection
  • the contacting step is not limited as long as the nucleic acid probe and the target substance in the sample can be specifically bound.
  • the nucleic acid probe and the sample may be simply mixed, or may be mixed in a medium such as water and a buffer.
  • the type of sample is not limited.
  • the sample may be a body fluid, cell or tissue isolated from a living body, or may be a purified and / or isolated target substance.
  • body fluids include blood, sweat, saliva, milk, urine, and semen.
  • Examples of cells include peripheral blood cells, lymph and tissue fluids containing cells, hair matrix cells, oral cells, nasal cells, intestinal cells. , Vaginal cells, mucosal cells, sputum (which may include alveolar cells or pneumohepatic cells).
  • tissues include lesion sites such as lymph nodes, bone marrow, spleen, thymus, and liver. For example, biopsy samples of these tissues can be used.
  • the insertion step is not limited as long as the intercalator can be inserted into the nucleic acid probe, and the intercalator and the sample may be simply mixed, or may be mixed in a medium such as water and a buffer. Further, when the target substance is present on the gel, such as a gel shift assay, a solution containing an intercalator may be permeated into the gel. The details of the intercalator are the same as described in “1. Nucleic acid probe for detecting target substance”, and therefore the description is omitted.
  • the nucleic acid probe is detected based on a signal such as fluorescence from the intercalator. If a nucleic acid probe bound to the target substance is detected in this step, it can be estimated that the target substance is present in the sample.
  • the detection step is by gel shift assay or flow cytometry.
  • Gel shift assay (EMSA: Electrophoresis Mobility Shift Assay, also called electrophoretic mobility assay) means that the total molecular weight of nucleic acid molecules bound to proteins is larger than that of nucleic acid molecules alone, and the mobility is different. This is a technique for separating and visualizing nucleic acid molecules bound to proteins using them.
  • Flow cytometry refers to a technique for measuring the number of cells and particles suspended in a liquid, and individual physical, chemical, and biological properties. The flow cytometry is preferably FACS (fluorescence-activated cell sorting) for analyzing cells.
  • the step of separating the nucleic acid probe bound to the target substance and the nucleic acid probe not bound to the target substance before the post-insertion step after the contacting step or before the detection step after the insertion step (separation step) Optionally included.
  • the separation step may be any of known methods such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, chromatography such as HPLC, ammonium sulfate fractionation, ultrafiltration, and immunoadsorption. It can carry out by using one or these in combination of 2 or more.
  • the separation step either the sample or the nucleic acid probe is immobilized on a solid support, and a sample in which no binding or non-specific binding has occurred is washed with a solution such as water and a buffer. It can also be done.
  • the present invention relates to a method for measuring the amount of a target substance using the nucleic acid probe described in “1.
  • Target substance detection nucleic acid probe in addition to the contact step, the insertion step, and any separation step, the step of measuring the amount of the nucleic acid probe bound to the target substance based on the intercalator (measurement step), and the nucleic acid probe bound to the target substance A step of estimating the amount of the target substance based on the amount of (estimation step). Since the contact step, the insertion step, and the optional separation step are the same as those described in the method for detecting the presence or absence of the target substance, description thereof is omitted here.
  • the measurement process can be performed in the same manner as the above detection process. That is, the amount of the nucleic acid probe can be measured based on a signal such as fluorescence of the intercalator. In this step, the amount of the nucleic acid probe is expressed as the signal intensity of the intercalator. In one embodiment, the measuring step is by gel shift assay or flow cytometry.
  • the amount of the target substance contained in the sample is estimated based on the amount of nucleic acid probe (intercalator signal intensity). For example, a correlation between a known amount of a target substance (reference) and signal intensity is clarified in advance, a calibration curve or the like is prepared, and based on this, the amount of the target substance can be estimated from the signal intensity.
  • the present invention uses the ELISA sandwich method to detect the presence or amount of a target substance using the nucleic acid probe of the present invention instead of the secondary antibody.
  • a target substance or sample is immobilized on a support using a primary antibody, a nucleic acid probe is added thereto, a nucleic acid probe that has not bound to the target substance or sample is removed, and the target substance is based on an intercalator. The presence or absence of a nucleic acid probe bound to can be detected or measured.
  • the method for detecting the presence or absence of the target substance and the method for measuring the amount of the target substance of the present invention may be performed on one target substance or sample, or may be performed on a plurality of target substances or samples. Also good.
  • the method of the present invention can be applied to a protein array or chemical array in which proteins or chemical substances are arranged.
  • Example 1 Synthesis of nucleic acid sensing probe containing RNA aptamer and evaluation of binding property to target protein> [Materials and methods] (Plasmid construction) A plasmid pET28b-T7-S1mt encoding an RNA aptamer S1mt that specifically binds to streptavidin was constructed.
  • Electrophoresis was performed at room temperature (constant voltage 100 V, 30 minutes), and then immersed in an aqueous EtBr solution (1 / 10,000) for 30 minutes or more in order to stain the gel. After the staining, UV (362 nm) was irradiated with a handy UV lamp (Handy UV Lamp SLUV-6, AS ONE), and a gel containing the necessary band was cut out. The linear plasmid DNA was extracted from the excised gel using QIAquick Gel Extraction Kit (QIAGEN).
  • RNA aptamer S1mt (GGCCGCCGACCAGAAUCAUGCAAGUGCGUAAGAUAGUCGCGGGUCGGCGGCC (SEQ ID NO: 1)) was synthesized.
  • ssDNA SalI-T7-S1mt-BglII (TCGACTTAATACGACTCACTATAGGCCGCCGACCAGAATCATGCAAGTGCGTAAGATAGTCGCGGGTCGGCGGCCAAAAAA (SEQ ID NO: 2)) and its complementary strand ssDNA (10 M
  • T4 PNK buffer 2.5l ⁇ l
  • sterile water1 (17.5 ⁇ l)
  • T4 ⁇ Polynucleotide Kinase (1.5 ⁇ l) was added and incubated at 37 C for 45 minutes.
  • phosphorylated ssDNA and complementary strand ssDNA were mixed, incubated at 95 ° C. for 2.5 minutes, allowed to stand at room temperature for 30 minutes or more, and then slowly
  • E. coli DH5 ⁇ competent cell (TaKaRa, 5 ⁇ l) dissolved on an ice bath and the plasmid DNA constructed above (pET28b-T7-S1mt) were mixed on an ice bath, and 42 After heating for 1 minute in a water bath at 0 ° C., it was immediately returned to the ice bath and rapidly cooled. Then, SOC medium (TaKaRa, 200 ⁇ l) was added in a clean bench, and the cells were evenly spread on a LB plate medium containing Kanamycin using a cell scraper and incubated at 37 ° C. overnight.
  • SOC medium (TaKaRa, 200 ⁇ l) was added in a clean bench, and the cells were evenly spread on a LB plate medium containing Kanamycin using a cell scraper and incubated at 37 ° C. overnight.
  • miniprep was performed using QIA prep Spin Miniprep Kit (QIAGEN).
  • Escherichia coli cultured in the test was dispensed together with LB medium into three 1.5 ml sample tubes and centrifuged at 8,000 rpm for 2 minutes.
  • P1 buffer 250 ⁇ l was added, suspended and collected.
  • P2 buffer 250 ⁇ l
  • N3 buffer 350 ⁇ l
  • the mixture was centrifuged at 13,000 rpm for 10 minutes to precipitate impurities. Further, the supernatant was taken so as not to touch the pellet, transferred to a column, centrifuged at 13,000 rpm for 1 minute, and the filtrate was discarded. Subsequently, PE buffer (600 ⁇ l) was added to the column and washed by centrifugation at 13,000 rpm for 1 minute. The filtrate was discarded again and centrifuged at 13,000 rpm for 1 minute to remove the remaining PE buffer.
  • PE buffer 600 ⁇ l
  • a plasmid pET28b-T7-A8 encoding a sequence in which eight adenines were linked was constructed. Between the restriction enzyme sites SalI and BglII of this plasmid, the sequence of ssDNATCSalI-T7-A8-BglII (TCGACTTAATACGACTCACTATAGGCAAAAAAAA (SEQ ID NO: 3)) is inserted.
  • the synthesized template DNA was made up to 135 ⁇ l with sterilized water, 15 ⁇ l of 10 ⁇ x loading dye was added, and 150 ⁇ l was loaded on agarose gel (2% Sea Plaque® Agarose). Electrophoresis was performed at room temperature (constant voltage: 100 V, 40 minutes), and then immersed in an EtBr aqueous solution (1 / 10,000) for 30 minutes or longer to stain the gel. Then, UV (362 nm) was irradiated with a handy UV lamp (Handy UV Lamp SLUV-6, AS ONE), and a gel containing the necessary band was cut out. In addition, the DNA extraction operation from the cut out gel utilized QIAquick Gel Extraction Kit (QIAGEN).
  • QIAquick Gel Extraction Kit QIAquick Gel Extraction Kit
  • template DNA (1.8 ⁇ g) was sterilized water (25 ⁇ l), 5 x T7 Trans buffer (10 ⁇ l), rNTP (rATP , RCTP, rGTP, rUTP) (100 mM each, 10 ⁇ l) and T7 Enz. Mix RNA Polymerase (5 ⁇ l), and incubated at 37 ° C. for 2.5 hours. Thereafter, RQ1 RNase free DNase (5 ⁇ l) was added and further incubated at 37 ° C. for 1 hour.
  • Buffer RLT QIAGEN, 175 ⁇ l
  • ⁇ -mercaptoethanol 1.75 ⁇ l
  • ethanol 125 ⁇ l
  • the mixed solution was added to an RNase Spin Column (QIAGEN, hereinafter, column), centrifuged at 10,000 rpm for 1 minute, and the filtrate was discarded.
  • Buffer RPE QIAGEN, 500 ⁇ l was added to the column, and the operation of centrifuging at 10,000 rpm for 1 minute was repeated twice, and then the filtrate was discarded and centrifuged again at 10,000 rpm for 1 minute.
  • Plasmid pET28b-T7-S1mt Forward Primer fp-T7-pET28b (CTTAATACGACTCACTATAGG (SEQ ID NO: 4)
  • Reverse Primer rp-pET28b-200 (GTGCATGCAAGGAGATG (SEQ ID NO: 5))
  • the temperature and time of heat denaturation, annealing, and extension reaction of double-stranded DNA are set as [98 ° C, 10 sec. ⁇ 55 °C, 15 sec. ⁇ 68 °C, 15 sec]. 19 cycles were carried out as a cycle.
  • template DNA (2 ⁇ g) was sterilized in water (25 ⁇ l), 5 x T7 Trans buffer (10 ⁇ l), rNTP (rATP , RCTP, rGTP, rUTP) (100 M each, 10 ⁇ l), T7 Enz.
  • Mix RNA Polymerase (5 ⁇ l) were mixed and incubated at 37 ° C for 2.5 hours. Thereafter, RQ1 RNase free DNase (5 ⁇ l) was added and further incubated at 37 ° C. for 1 hour.
  • Buffer RLT QIAGEN, 175 ⁇ l
  • ⁇ -mercaptoethanol 1.75 ⁇ l
  • ethanol 125 ⁇ l
  • Buffer RPE QIAGEN, 500 ⁇ l
  • the operation of centrifuging at 10,000 rpm for 1 minute was repeated twice, and then the filtrate was discarded and centrifuged again at 10,000 rpm for 1 minute.
  • the column was set in an RNase free sample tube, sterilized water (55 ⁇ l) was added, left to stand for 2.5 hours, and centrifuged at 10,000 rpm for 2 minutes to elute and collect purified RNA.
  • the plasmid DNA and primer pairs used for the above synthesis are described below.
  • Plasmid pET28b-T7-S1mt Forward Primer fp-after-RNA (GATCTCGATCCTCTACG (SEQ ID NO: 6)
  • Reverse Primer T7-rp-pET28b-200 TTAATACGACTCACTATAGGTGCATGCAAGGAGATG (SEQ ID NO: 7)
  • S1mt-dsRNA (200) and C-ssRNA (200) purified above were hybridized to synthesize S1mt-dsRNA (double stranded RNA: double-stranded RNA, hereinafter simply referred to as dsRNA) (200). It was.
  • MOPS buffer (18.3 ⁇ l) are mixed, incubated at 95 C for 2.5 minutes, and then The mixture was allowed to stand at room temperature for 30 minutes or more and allowed to cool slowly to allow hybridization.
  • production of S1mt-dsRNA (200) was confirmed by electrophoresis.
  • S1mt-dsRNA (400) with an extended double stranded site was synthesized.
  • S1mt-ssRNA (400) SEQ ID NO: 14
  • the template DNA was synthesized by PCR.
  • sterile water (336 ⁇ l), 5) x GXL buffer (120 ⁇ l), 2.5 mM dNTP (48 ⁇ l), 10 ⁇ M Forward Primer (12 ⁇ l), 10 ⁇ M Reverse Primer (12 ⁇ l), 1 ng / ⁇ l plasmid
  • Prime STAR GXL
  • 25 ⁇ l was dispensed into a PCR tube, and PCR was performed.
  • PCR conditions the temperature and time of heat denaturation, annealing, and extension reaction of double-stranded DNA were set as [98 ° C, 10 sec. ⁇ 52 °C, 15 sec. ⁇ 68 °C, 25 sec].
  • 20 cycles 20 cycles were performed.
  • the synthesized template DNA was made up to 135 ⁇ l with sterile water, 15 ⁇ l of 10 ⁇ x loading dye was added, and 150 ⁇ l loaded on an agarose gel (2% Sea Plaque Agarose). Electrophoresis was performed at room temperature (constant voltage: 100 V, 40 minutes), and then immersed in an aqueous EtBr solution (1 / 10,000) for 30 minutes or more to stain the gel. Then, UV (362 nm) was irradiated with a handy-type UV lamp (Handy UV Lamp SLUV-6, AS ONE), and a gel containing a necessary band was cut out. In addition, the DNA extraction operation from the cut out gel utilized QIAquick Gel Extraction Kit (QIAGEN).
  • QIAquick Gel Extraction Kit QIAquick Gel Extraction Kit
  • template DNA 1.3 ⁇ g was sterilized with water (12.5 ⁇ l), 5 x T7 Trans buffer (5 ⁇ l), rNTP (rATP, rCTP, rGTP, rUTP) (100 mM each, 5 ⁇ l) and T7 Enz.
  • rNTP rATP, rCTP, rGTP, rUTP
  • the mixed solution was added to an RNase Spin Column (QIAGEN, hereinafter, column), centrifuged at 10,000 rpm for 1 minute, and the filtrate was discarded.
  • Buffer RPE QIAGEN, 500 ⁇ l was added to the column, and the operation of centrifuging at 10,000 rpm for 1 minute was repeated twice, and then the filtrate was discarded and centrifuged again at 10,000 rpm for 1 minute.
  • the column was set in an RNase free sample tube, sterilized water (55 ⁇ l) was added, and the mixture was allowed to stand for 2.5 hours, and then centrifuged at 10,000 rpm for 2 minutes to elute and collect the purified RNA.
  • Plasmid pET28b-T7-S1mt Forward Primer fp-T7-pET28b (SEQ ID NO: 4)
  • Reverse Primer rp-pET28b-400 (GCGACATCGTATAACGTTAC (SEQ ID NO: 8))
  • sterile water (336 ⁇ l), 5) x GXL buffer (120 ⁇ l), 2.5 mM dNTP (48 ⁇ l), 10 ⁇ M Forward Primer (12 ⁇ l), 10 ⁇ M Reverse Primer (12 ⁇ l), 1 ng / ⁇ l plasmid DNA (60 ⁇ l) was mixed, then Prime STAR GXL (12 ⁇ l) was added, and 25 ⁇ l was dispensed into a PCR tube, and PCR was performed.
  • the temperature and time of heat denaturation, annealing, and extension reaction of double-stranded DNA are set as [98 ° C, 10 sec. ⁇ 55 °C, 15 sec. ⁇ 68 °C, 25 sec]. 19 cycles were carried out as a cycle.
  • the synthesized template DNA was made up to 135 ⁇ l with sterile water, 15 ⁇ l of 10 ⁇ x loading dye was added, and 150 ⁇ l was loaded onto agarose gel (2% Sea Plaque® Agarose). Electrophoresis was performed at room temperature (constant voltage: 100 V, 40 minutes), and then immersed in an aqueous EtBr solution (1 / 10,000) for 30 minutes or more to stain the gel. Then, UV (362 nm) was irradiated with a handy UV lamp (Handy UV Lamp SLUV-6, AS ONE), and a gel containing the necessary band was cut out. In addition, DNA extraction operation from the excised gel utilized QIAquickquiGel Extraction Kit (QIAGEN).
  • QIAquickquiGel Extraction Kit QIAquickquiGel Extraction Kit
  • template DNA (2.3 ⁇ g) was sterilized with water (25 ⁇ l), 5 x T7 Trans buffer (10 ⁇ l), rNTP (rATP , RCTP, rGTP, rUTP) (100 mM each, 10 ⁇ l) and T7 Enz.
  • Mix RNA Polymerase (5 ⁇ l) were mixed and incubated at 37 ° C. for 3 hours. Thereafter, RQ1 RNase free DNase (5 ⁇ l) was added and further incubated at 37 ° C. for 1 hour.
  • Buffer RLT QIAGEN, 175 ⁇ l
  • ⁇ -mercaptoethanol 1.75 ⁇ l
  • ethanol 125 ⁇ l
  • Plasmid pET28b-T7-S1mt Forward Primer fp-after-RNA SEQ ID NO: 6
  • Reverse Primer T7-rp-pET28b-400 TTAATACGACTCACTATAGGCGACATCGTATAACGTTAC (SEQ ID NO: 9)
  • S1mt-dsRNA (400) was synthesized by hybridizing S1mt-ssRNA (400) and C-ssRNA (400) purified above.
  • S1mt-ssRNA (400) (16.27 ⁇ M, 1.23 ⁇ l)
  • C-ssRNA (400) 21.76 ⁇ M, 0.83 ⁇ l
  • Tris-HCl buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl 2 , 17.9 ⁇ l
  • bb-ds was confirmed by electrophoresis.
  • A8-dsRNA (200) and A8-dsRNA (400) First, in order to synthesize A8-ssRNA (200) (SEQ ID NO: 16) in a test tube, template DNA was synthesized. Sterile water (168 ⁇ l), 5 x GXL buffer (60 ⁇ l), 2.5 mM dNTP (24 ⁇ l), 10 ⁇ M Forward Primer (6 ⁇ l), 10 ⁇ M Revers Primer (6 ⁇ l), 1 ng / ⁇ l plasmid DNA (30 After mixing ( ⁇ l), Prime STAR GXL (6 ⁇ l) was added, 25 ⁇ l was dispensed into PCR tubes, and PCR was performed.
  • PCR conditions set the temperature and time of heat denaturation, annealing, and extension reaction of double-stranded DNA as [98 °C, 10 sec. ⁇ 55 °C, 15 sec. ⁇ 68 °C, 15 sec.]. 20 cycles were carried out as one cycle.
  • template DNA (1 ⁇ g) was sterilized with water (12.5 ⁇ l), 5 x T7 Trans buffer (5 ⁇ l), rNTP (rATP , RCTP, rGTP, rUTP) (100 M each, 5 ⁇ l), T7 Enz.
  • Mix RNA Polymerase (2.5 ⁇ l) were mixed and incubated at 37 ° C for 2.5 hours. Thereafter, RQ1 RNase free DNase (2.5 ⁇ l) was added and further incubated at 37 ° C for 1 hour.
  • Buffer RLT QIAGEN, 87.5 ⁇ l
  • ⁇ -mercaptoethanol 0.875 ⁇ l
  • ethanol 62.5 ⁇ l
  • the mixture was added to an RNase spin column (QIAGEN, hereinafter referred to as column), centrifuged at 10,000 rpm for 1 minute, and the filtrate was discarded.
  • Buffer RPE QIAGEN, 500 ⁇ l was added to the column, and the operation of centrifuging at 10,000 rpm for 1 minute was repeated twice, and then the filtrate was discarded and centrifuged again at 10,000 rpm for 1 minute.
  • A8-ssRNA (200) (25.2 ⁇ M, 0.79 ⁇ l), C-ssRNA (200) (25.7 ⁇ M, 0.78 ⁇ l), MOPS buffer (18 ⁇ l), sterile waterpox (0.43 ⁇ l), 95 Incubated at ° C for 2.5 minutes.
  • A8-dsRNA (200) was synthesized by allowing hybridization to proceed by allowing it to stand at room temperature for 30 minutes or more and allowing it to cool slowly.
  • A8-dsRNA (400) was synthesized by hybridizing A8-ssRNA (400) (SEQ ID NO: 17) and C-ssRNA (400) synthesized in Example 1 according to the synthesis method of A8-dsRNA (200). (However, the template DNA necessary for the synthesis of A8-ssRNA (400) was synthesized by PCR using plasmid pET28b-T7-A8, Forward Primer fp-T7-pET28b, Reverse Primer rp-pET28b-400. did).
  • Streptavidin (1 ⁇ M, 3 ⁇ l) is mixed with S1mt-dsRNA (200) or S1mt-dsRNA (400) (0.2 ⁇ M, 1.5 ⁇ l), Tris-HCl buffer (4.5 ⁇ l) for 15 minutes at room temperature. Incubated above.
  • S1mt-dsRNA (200) or S1mt-dsRNA (400) 0.2 ⁇ M, 1.5 ⁇ l
  • Tris-HCl buffer 4.5 ⁇ l
  • those using hDHFR (ATGEN) instead of streptavidin
  • A8-dsRNA (200) or A8-dsRNA (400) instead of S1mt-dsRNA (200) or S1mt-dsRNA (400)
  • the ones used were also prepared. Thereafter, glycerol (1 ⁇ l) was added, and 10 ⁇ l of each was loaded onto a 4% polyacrylamide gel.
  • Electrophoresis was performed under ice-cooling (constant voltage: 130 V, 73 minutes), and then immersed in an EtBr aqueous solution (1 / 10,000) for 15 minutes or longer for gel staining.
  • the band pattern of the nucleic acid sensing probe was evaluated with a bioimager (BioDoc-IT Imaging System, UVP).
  • sequence of ssDNA SalI-T7-boxB-BglII (TCGACTTAATACGACTCACTATAGGCCCTGAAAAAGGGCCAAAAAAAA (SEQ ID NO: 10)) is inserted between the restriction enzyme sites SalI and BglII of this plasmid.
  • a plasmid pET28b-T7-boxB-A8-boxB encoding a sequence in which two boxB sequences were linked via a sequence in which eight adenines were linked was constructed.
  • the sequence of ssDNABSalI-T7-boxB-A8-boxB-BglII (TCGACTTAATACGACTCACTATAGGCCCTGAAAAAGGGCCAAAAAAAAGGCCCTGAAAAAGGGCCAAAAAAAA (SEQ ID NO: 11)) is inserted.
  • b-ssRNA (200) in a test tube using plasmid DNA encoding an RNA motif having a boxB sequence as a template sterilized water (168) ⁇ l), 5 x GXL buffer (60 ⁇ l), 2.5 mM dNTP ( 24 ⁇ l), 10 ⁇ M Forward Primer (6 ⁇ l), 10 ⁇ M Reverse Primer (6 ⁇ l), 1 ng / ⁇ l Plasmid DNA (30l ⁇ l), then Prime STAR GXL (6 ⁇ l) PCR was carried out by dispensing 25 ⁇ l aliquots into each.
  • the temperature and time of heat denaturation, annealing, and extension reaction of double-stranded DNA are set as [98 ° C, 10 sec. ⁇ 55 °C, 15 sec. ⁇ 68 °C, 15 sec]. 20 cycles were carried out as a cycle.
  • template DNA (1 ⁇ g) was sterilized with water (12.5 ⁇ l), 5 x T7 Trans buffer (5 ⁇ l), rNTP (rATP , RCTP, rGTP, rUTP) (100 M each, 5 ⁇ l), T7 Enz.
  • Mix RNA Polymerase (2.5 ⁇ l) were mixed and incubated at 37 ° C for 2.5 hours. Then, RQ1 RNase free DNase (5 ⁇ l) was added and further incubated at 37 ° C for 1 hour.
  • Buffer RLT QIAGEN, 87.5 ⁇ l
  • ⁇ -mercaptoethanol 0.875 ⁇ l
  • ethanol 62.5 ⁇ l
  • Buffer RPE QIAGEN, 500 ⁇ l
  • template DNA was synthesized by PCR.
  • sterilized water 168 ⁇ l
  • 5 x GXL buffer 60 ⁇ l
  • 2.5 mM dNTP 24 ⁇ l
  • 10 ⁇ M Forward Primer 6 ⁇ l
  • 10 ⁇ M Reverse Primer 6 ⁇ l
  • 1 ⁇ ng / ⁇ l plasmid DNA After mixing (30 ⁇ l), Prime STAR GXL (6 ⁇ l) was added, and 25 ⁇ l each was dispensed into a PCR tube, and PCR was performed.
  • the temperature and time of heat denaturation, annealing, and extension reaction of double-stranded DNA are set as [98 ° C, 10 sec. ⁇ 55 °C, 15 sec. ⁇ 68 °C, 15 sec].
  • 22 cycles were carried out.
  • template DNA (1 ⁇ g) was sterilized with water (12.5 ⁇ l), 5 x T7 Trans buffer (5 ⁇ l), rNTP (rATP , RCTP, rGTP, rUTP) (100 M each, 5 ⁇ l), T7 Enz.
  • Mix RNA Polymerase (2.5 ⁇ l) were mixed and incubated at 37 ° C for 2.5 hours. Then, RQ1 RNase free DNase (5 ⁇ l) was added and further incubated at 37 ° C for 1 hour.
  • Buffer RLT QIAGEN, 87.5 ⁇ l
  • ⁇ -mercaptoethanol 0.875 ⁇ l
  • ethanol 62.5 ⁇ l
  • Buffer RPE QIAGEN, 500 ⁇ l
  • RNA was set in an RNase free sample tube, sterilized water (55 ⁇ l) was added, allowed to stand for 2.5 hours, and centrifuged at 10,000 rpm for 2 minutes to elute and collect purified RNA.
  • the plasmid DNA and primer pair used for the synthesis of the above bb-ssRNA (200) are shown below. Plasmid pET28b-T7-boxB-A8-boxB Forward Primer fp-T7-pET28b (SEQ ID NO: 4) Reverse Primer rp-pET28b-200 (SEQ ID NO: 5)
  • bb-ssRNA (200) (22.8 ⁇ M, 0.9 ⁇ l), C-ssRNA (200) (28.9. ⁇ M, 0.69 ⁇ l), MOPS buffer (18.43 ⁇ l) are mixed and incubated at 95 ° C for 2.5 minutes. Hybridization was carried out by leaving it to stand at room temperature for 30 minutes or more and slowly cooling it.
  • the base plasmid used here is pET32c (Novagen), and a sequence encoding the target protein is inserted between the restriction enzyme sites NdeI and HindIII of pET32c-Bap-GS2-Halo-F ( SEQ ID NO: 21). On the other hand, a sequence encoding the target protein is inserted between the restriction enzyme sites NdeI and HindIII of pET32c-Bap-GS2-Bap-GS2-Halo-F (SEQ ID NO: 22).
  • template DNA was synthesized by PCR to synthesize mRNA encoding the target protein Bap-GS2-Halo-F.
  • the PCR here was performed based on the template DNA synthesis procedure used to obtain the S1mt-ssRNA.
  • plasmid pET32c-Bap-GS2-Halo-F, ForwardForPrimer (TAATACGACTCACTATAGGG, SEQ ID NO: 23), Reverse Primer (TCATTACTTGTCGTCATCGTC, SEQ ID NO: 24) are used as templates.
  • template DNA was synthesized by PCR in order to synthesize mRNA encoding the target protein Bap-GS2-Bap-GS2-Halo-F.
  • PCR was carried out using plasmid pET32c-Bap-GS2-Bap-GS2-Halo-F, Forward ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ Primer (same as above), Reverse Primer (same as above) as a template.
  • mRNA encoding Bap-GS2-Halo-F and Bap-GS2-Bap-GS2-Halo-F was synthesized by in vitro transcription of the template DNA obtained above. Transcription here was performed based on the procedure for synthesizing S1mt-ssRNA.
  • Bap-GS2-Halo-F and Bap-GS2-Bap-GS2-Halo-F were synthesized in vitro using a cell-free protein synthesis kit PUREfrex 1.0 (Gen Frontier) and various mRNA (12 pmol).
  • the solution (5 ⁇ l), Solution I (6.25 ⁇ l), Solution II (0.625 ⁇ l), Solution III (0.625 ⁇ l) were mixed and incubated at 37 ° C. for 2.5 hours.
  • the translation solution of Bap-GS2-Halo-F (1.4 ⁇ l) was compared with b-dsRNA (0.2 ⁇ M, 1.5 ⁇ l) or A8-dsRNA (0.2 ⁇ M, 1.5 ⁇ l).
  • MOPS buffer (20 mM mMPS, 100 mM NaCl) were mixed. Then, incubation was performed at room temperature for 15 minutes or more. Thereafter, glycerol (1 ⁇ l) was added, and 10 ⁇ l ⁇ l was loaded onto a 4% polyacrylamide gel.
  • Electrophoresis was performed at room temperature (constant voltage: 130 V, 45 min), and then immersed in EtBr aqueous solution (1 / 10,000) for 15 min or longer as gel staining.
  • the band pattern of the nucleic acid sensing probe was evaluated using a bioimager (BioDoc-IT Imaging System, UVP). Further, when bb-dsRNA (200) was used, gel shift assay was performed in the same procedure as described above.
  • bb-dsRNA (400) (Synthesis of bb-dsRNA (400) and evaluation of target binding by EMSA)
  • a bb-dsRNA (400) with an extended double-stranded RNA site was synthesized and the same EMSA was performed.
  • the synthesis of bb-dsRNA (400) is performed by hybridizing bb-ssRNA (400) (SEQ ID NO: 20) and C-ssRNA (400) synthesized in Example 1 according to the synthesis method of bb-dsRNA (200).
  • the template DNA necessary for the synthesis of bb-ssRNA (400) is plasmid pET28b-T7-boxB-A8-boxB, Forward Primer fp-T7-pET28b, Reverse Primer rp-pET28b-400. Synthesized by PCR).
  • the amount of the translation solution of Bap-GS2-Bap-GS2-Halo-F mixed with bb-dsRNA (400) was 2.7 ⁇ l and 1.4 ⁇ l, and gel shift assay was performed in the same procedure as above. .
  • the cell-free protein synthesis kit PUREfrex® 1.0 used this time contains 20 amino acids, 20 aminoacyl tRNA synthetases, and a number of translation factors such as ribosomes. Therefore, it is clear that it is possible to detect only the target protein simply and quickly by the specific interaction between the nucleic acid sensing probe presenting the RNA motif and the peptide even in a solution containing multiple components. It became.
  • a probe capable of detecting a target substance with high sensitivity is provided, and a simple and highly sensitive method for detecting and / or measuring a target substance using the probe can be provided.
  • the detection portion of the probe can be designed, for example, as an aptamer capable of binding to a specific substance, and a probe capable of detecting a specific substance with high sensitivity is provided. Therefore, the influence on the industry of the present invention is great.

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Abstract

La présente invention concerne le problème consistant à se munir : d'une sonde à acide nucléique qui peut être préparée facilement et qui peut être détectée avec une sensibilité élevée ; et autres. La présente invention concerne également le problème consistant à se munir : d'un procédé pour déterminer la présence ou l'absence d'une substance cible ou mesurer la quantité de la substance cible à l'aide de la molécule d'acide nucléique ; et autres. La présente invention concerne : une sonde d'acide nucléique pour détecter une substance cible, qui contient une première molécule d'acide nucléique simple brin et une seconde molécule d'acide nucléique simple brin, la première molécule d'acide nucléique simple brin contenant une région de liaison cible capable de se lier spécifiquement à la substance cible et une première région d'acide nucléique simple brin liée à l'une ou l'autre des extrémités de la région de liaison cible, la seconde molécule d'acide nucléique simple brin contenant une seconde région d'acide nucléique simple brin contenant les bases complémentaires de la séquence nucléotidique pour la première région d'acide nucléique simple brin, et la première région d'acide nucléique simple brin et la seconde région d'acide nucléique simple brin étant appariées par bases l'une avec l'autre pour former une région de détection dans laquelle un intercalaire peut être inséré ; un procédé de détermination de la présence ou de l'absence d'une substance cible ou de mesure de la quantité de la substance cible à l'aide de la sonde ; et autres.
PCT/JP2018/005032 2017-02-14 2018-02-14 Sonde à acide nucléique pour détecter une substance cible WO2018151138A1 (fr)

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Citations (5)

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JP2005257667A (ja) * 2004-02-09 2005-09-22 Sony Corp 物質間の相互作用を検出する検出表面と該検出表面を用いるセンサチップとセンサ装置及び検出方法
JP2005304489A (ja) * 2004-03-24 2005-11-04 Sysmex Corp 標的物質検出用プローブセット及び標的物質検出方法。
WO2013014843A1 (fr) * 2011-07-25 2013-01-31 日本電気株式会社 Procédé de détection d'une substance cible, puce de détection, et appareil de détection
WO2016028223A1 (fr) * 2014-08-22 2016-02-25 Agency For Science, Technology And Research Aptamères
WO2016159197A1 (fr) * 2015-03-30 2016-10-06 京セラ株式会社 Aptamère arn et capteur l'utilisant

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JP2005257667A (ja) * 2004-02-09 2005-09-22 Sony Corp 物質間の相互作用を検出する検出表面と該検出表面を用いるセンサチップとセンサ装置及び検出方法
JP2005304489A (ja) * 2004-03-24 2005-11-04 Sysmex Corp 標的物質検出用プローブセット及び標的物質検出方法。
WO2013014843A1 (fr) * 2011-07-25 2013-01-31 日本電気株式会社 Procédé de détection d'une substance cible, puce de détection, et appareil de détection
WO2016028223A1 (fr) * 2014-08-22 2016-02-25 Agency For Science, Technology And Research Aptamères
WO2016159197A1 (fr) * 2015-03-30 2016-10-06 京セラ株式会社 Aptamère arn et capteur l'utilisant

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HE, X. ET AL.: "Aptamer-fluorescent silica nanoparticles bioconjugates based dual-color flow cytometry for specific detection of Staphylococcus aureus", J. BIOMED. NANOTECHNOL., vol. 10, no. 7, July 2014 (2014-07-01), pages 1359 - 1368, Retrieved from the Internet <URL:https://doi.org/10.1166/jbn.2014.1828> *

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