WO2008020589A1 - Brin d'acide nucléique utile pour détecter une substance et procédé de détection correspondant - Google Patents
Brin d'acide nucléique utile pour détecter une substance et procédé de détection correspondant Download PDFInfo
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- WO2008020589A1 WO2008020589A1 PCT/JP2007/065844 JP2007065844W WO2008020589A1 WO 2008020589 A1 WO2008020589 A1 WO 2008020589A1 JP 2007065844 W JP2007065844 W JP 2007065844W WO 2008020589 A1 WO2008020589 A1 WO 2008020589A1
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6823—Release of bound markers
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- 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
- C12Q1/6804—Nucleic acid analysis using immunogens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- 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
- C12Q2521/00—Reaction characterised by the enzymatic activity
- C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
- C12Q2521/301—Endonuclease
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- 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
- C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
- C12Q2525/10—Modifications characterised by
- C12Q2525/119—Modifications characterised by incorporating abasic sites
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- 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
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- 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
- C12Q2565/1015—Interaction between at least two labels labels being on the same oligonucleotide
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Definitions
- Nucleic acid strands useful for detecting substances and methods are provided.
- the present invention relates to a nucleic acid chain useful for detecting a substance and the related technology. More specifically
- the present invention also relates to a nucleic acid chain in which an enzyme cleavage site is present in the base sequence portion between the fluorescent substance and the quencher substance, and a method using the nucleic acid chain.
- a technique for detecting the presence or reaction of a substance by detecting fluorescence emitted from the substance is known and widely used in various sensor techniques.
- a probe substance capable of specifically interacting with the substance present in the reaction field is introduced into the reaction field, and fluorescence previously labeled on the probe substance is converted into the reaction field.
- a technique for confirming whether or not it can be detected from the field is known.
- a method of introducing a fluorescently labeled nucleic acid chain toward a nucleic acid chain immobilized on the surface of a substrate (chip) and detecting the presence or absence of hybridization between the two nucleic acid chains by fluorescence detection is the method of a DNA chip. It is a conventional technique.
- intercalator when single-stranded nucleic acids existing in a reaction field are hybridized, a substance that specifically binds to its complementary binding portion and emits fluorescence, a so-called "intercalator”. Is also known. This intercalator has the advantage that it saves the trouble of pre-labeling the fluorescent substance on the nucleic acid chain.
- FRET Fluorescence Resonance Energy Transfer
- bioluminescence resonance energy transfer is a biophysical method capable of directly detecting protein-protein interactions.
- This “BRET” is a process found in marine organisms such as the jellyfish Aequore a victoria Renilla reniformis. It is possible to detect the energy release of the acceptor protein with fluorescence. Therefore, BRET can be a useful technique for detecting protein-protein interactions.
- a generally performed technique is to improve the detection sensitivity by amplifying the trace substance.
- a technique for amplifying a very small amount of nucleic acid chain to be detected by a PCR (polymerase chain reaction) method is widely used!
- the main object of the present invention is to provide a technique that can detect a trace amount substance with high sensitivity without performing amplification of the trace quantity substance.
- a fluorescent substance that can be a donor of resonance excitation energy, a quencher substance that exists at a position where the resonance excitation energy can be received, and the quencher substance
- a nucleic acid chain for detection having at least a nucleic acid chain portion located between the fluorescent substances and having an enzyme cleavage site cleaved by an endonuclease.
- nucleic acid chain means a polymer (nucleotide chain) of a phosphate ester of a nucleoside in which a purine or pyrimidine base and a sugar are glycosidically linked, and includes an oligonucleotide, polynucleotide, purine nucleotide and pyrimidine nucleoside containing probe DNA. It includes a wide range of DNAs (including full-length or fragments thereof), cDNA obtained by reverse transcription (c-probe DNA), and RNA.
- Endonuclease is a general term for nucleolytic enzymes that cleave the inside of a nucleic acid chain.
- the present enzyme is used alone or in combination with an enzyme that exerts another cooperative action.
- a “detection nucleic acid chain” is a nucleic acid chain used for the purpose of detecting a predetermined substance, reaction (including chemical bonds, interactions, etc.), structure, etc., and is sometimes called a probe nucleic acid chain. This is consistent with the concept.
- Examples of the enzyme cleavage site present in the nucleic acid chain part constituting the nucleic acid chain for detection according to the present invention include, for example, a base missing site (AP site that can be cleaved by a double-strand-specific AP-endonuclease. ) Power to raise S.
- a base missing site AP site that can be cleaved by a double-strand-specific AP-endonuclease.
- the “AP site (Apurinic / Apyrimidinic site)” means a site where a base is missing (dropped) in a nucleic acid chain! /
- AP-endonuclease is an enzyme having an AP lyase activity that inserts a nick (nick) in the nucleic acid chain.
- Endonucleas e VI-Endonuclease IV (cutting the 5 'side of the AP site), Endonuclease ⁇ (cutting the 3' side of the heel site 13 ⁇ 4, Endonuclease SI, Endonuclease VIII, Pormamidopynmidine-DNA-glycosyl ase (Fpg), OGG1, etc.
- Particularly useful AP-endonuclease in the present invention is to specifically recognize a double strand and one nucleic acid strand having a base deletion site to the base. It is an enzyme that cleaves at the missing site.
- the number of bases (number of nucleic acid molecules) of the nucleic acid chain according to the present invention is not particularly limited.
- it is an oligonucleotide chain
- the use of the nucleic acid chain is not particularly limited.
- it can be used as a probe for detecting a target substance (substance for detection).
- the oligonucleotide chain means a nucleotide polymer of about several tens of bases, for example, about 15 to 30 bases.
- the nucleic acid chain portion may be, for example, a protein having a response base sequence region that binds to a predetermined protein.
- the protein is a protein having a function of binding to a nucleic acid chain, and an example thereof is a transcription factor. .
- the present invention provides (1) a fluorescent substance that can be a donor of resonance excitation energy, and a position where the resonance excitation energy can be received.
- a fluorescent substance that can be a donor of resonance excitation energy, and a position where the resonance excitation energy can be received.
- the present invention provides a method for using a nucleic acid chain that at least carries out the procedure for amplifying the fluorescence of the fluorescent substance by dissociating it into a single strand, and the procedures (1) and (2).
- the "complementary strand formation reaction” is a so-called hybridization between nucleic acids (nucleotide strands), and widely includes DNA-DNA, DNA-RNA, RNA-RNA complementary binding, and the like.
- the detection nucleic acid strand in a fluorescence-reduced or quenched state is formed into a double strand with a complementary nucleic acid strand by the action of a quencher substance.
- the detection nucleic acid strand is cleaved at the endonuclease cleavage site and dissociated into single strands by the action of a double strand-specific endonuclease.
- the nucleic acid strand for detection is fragmented and released into a nucleic acid chain on the fluorescent substance side and a nucleic acid chain on the single substance side, so that the fluorescent substance is at a distance from the quencher substance.
- the fluorescence will be amplified (without the influence of the Taentia substance).
- the detection nucleic acid strand before endonuclease cleavage is not more than an upper limit temperature at which the complementary strand formation reaction can proceed, and the detection nucleic acid strand fragment after endonuclease cleavage is complementary strand formation.
- the reaction can be carried out under a temperature condition higher than the temperature at which the reaction cannot be maintained or proceeded.
- the detection nucleic acid strand formed a double strand (complementary strand), ie, the probe nucleic acid
- the target nucleic acid strand that is complementary to the strand exists, that there is a biological substance other than the nucleic acid, that there is a chemical or structural change in the biological substance, and that there is a drug candidate substance. , Etc. Power to know.
- the trace amount is detected. It can be detected with high sensitivity without amplification of the substance.
- FIG. 1 is a diagram for explaining the concept of a nucleic acid strand for detection according to the present invention and an example embodiment.
- FIG. 2 is a diagram showing an embodiment in which both a fluorescent substance (F) and a quencher substance (Q) are bound to a midway portion of the nucleic acid chain among the nucleic acid chains for detection.
- Fig. 3 is a view showing another embodiment example in which the fluorescent substance (F) is bound to the terminal portion and one quencher substance (Q) is bound to the intermediate portion of the detection nucleic acid chain.
- Fig. 4 is a view showing still another embodiment in which the fluorescent substance (F) is bound to the middle part and one quencher substance (Q) is bound to the terminal part of the nucleic acid chain for detection. .
- FIG. 5 is a diagram showing the function of the detection nucleic acid strand (P) according to the present invention and a typical use example thereof.
- FIG. 6 is a diagram schematically showing how a cut is formed in AP site X by AP-endonuclease E.
- FIG. 7 shows that the nucleic acid strand fragments (P, P) for detection cleaved by AP-endonuclease (E) dissociate from the nucleic acid strand (T) under the appropriate temperature condition (t).
- reaction chain (R) as a chain
- FIG. 8 is a diagram showing a basic flow of “cycle reaction” by the nucleic acid strand for detection P according to the present invention.
- Fig. 9 shows detection nucleic acid chains (P) that have not been cleaved and nucleic acids for detection (that have been cleaved by AP-endonuclease E) due to changes in the temperature conditions of the reaction field (R). It is a reference figure which shows the mode of the double strand formation or dissociation of a chain
- FIG. 10 is a schematic diagram showing the concept of a first applied method example using the detection nucleic acid strand (P) according to the present invention.
- FIG. 11 is a schematic diagram showing the concept of a second applied method example using the detection nucleic acid strand (P) according to the present invention.
- FIG. 12 is a schematic diagram showing a concept of a third applied method example using the detection nucleic acid strand (P) according to the present invention.
- FIG. 13 is a schematic diagram showing the concept of a fourth applied method example using the detection nucleic acid strand (P) according to the present invention.
- FIG. 14 is a schematic diagram showing the concept of a fifth applied method example using the detection nucleic acid strand (P) according to the present invention.
- FIG. 15 is a schematic diagram showing the concept of a sixth applied method example using the detection nucleic acid strand (P) according to the present invention.
- FIG. 16 is a diagram showing the concept of the seventh applied method example using the detection nucleic acid strand (P) according to the present invention, in which a substance (for example, protein Dx) before structural change is detected.
- a substance for example, protein Dx
- FIG. 17 is a diagram showing the concept of the seventh applied method example, and is a schematic diagram in the case of detecting a substance after structural change (for example, protein Dy).
- FIG. 18 is a diagram showing the concept of the eighth application example, and is a schematic diagram showing an example in which the detection nucleic acid chain P is used as a DNA chip probe.
- Fig. 19 shows the result of experiment 1: reaction of lOOfmol target nucleic acid strand and 8 pmol of detection nucleic acid strand (having AP site) with AP-endonuclease to detect cleavage of detection nucleic acid strand It is a figure (photograph which shows the result of agarose electrophoresis) which shows.
- Fig. 20 is a diagram (Experimental Example 2: Fluorescence signal amplification detected over time) (Experimental axis: Fluorescence intensity, Horizontal axis: Time (minutes)).
- FIG. 21 is a graph (graph) obtained by plotting Experimental Example 2: Concentration of each target nucleic acid strand and initial reaction rate using “Line weaver Burk Plotj”.
- Fig. 22 shows the results of measurement of the increase in fluorescence of Experimental Example 3: Fluorescent Dye (FITC) over time, and plots the increase rate of fluorescence per unit time at each time (drawing cost)
- FITC Fluorescent Dye
- FIG. 23 is a diagram (drawing substitute graph) showing the result of detecting BMAL1-CLOCK complex by shaking the number of Hela cells (horizontal axis) in Experimental Example 3: Hela cells.
- FIG. 24 shows experimental example 4: Hela cells were stimulated with a culture solution (DMEM medium) containing 50 Horse Serum for 2 hours, and the force at the start of stimulation was 15 hours, 17.5 hours, 20 hours, 22
- Figure 5 shows the results of recovering cells after 5 hours and detecting the BMAL1-CLOCK protein complex (drawing substitute)
- FIG. 25 is a diagram (drawing substitute graph) showing the result of quantifying the amount of mRNA of Experimental Example 4: Perl.
- FIG. 26 is a diagram (diagram substitute for drawing) showing the results of quantifying the amount of mRNA of Experimental Example 4: Per2;
- Figs. 1 to 4 are diagrams for explaining the concept of a nucleic acid chain for detection according to the present invention and an embodiment.
- the detection nucleic acid chain P includes a fluorescent substance F that can be a donor of resonance excitation energy, a quencher substance Q that exists at a position where the resonance excitation energy can be received, and the quencher substance.
- fluorescent substance F is bound (labeled) to the T-end or ⁇ -end of a nucleic acid chain having a predetermined molecular length, and a single substance Q is bound to the end opposite to the fluorescent substance F.
- the entire nucleic acid chain is located between the fluorescent substance F and the quencher substance Q, so that the entire nucleic acid chain part corresponds to the nucleic acid chain part N in the present invention. (See Figure 1).
- the nucleic acid strand region is the nucleic acid strand portion N referred to in the present invention (see FIGS. 2 to 4).
- Fig. 2 shows an embodiment in which both the fluorescent substance F and the quencher substance Q are bound to an intermediate site of the nucleic acid chain
- Fig. 3 shows the fluorescent substance F as a terminal site and one quencher
- FIG. 4 shows another embodiment in which the substance Q is bonded to the intermediate site
- FIG. Still another embodiment example in which the single substance Q is bound to the terminal site is shown respectively.
- FIG. 5 is a diagram showing the function of the detection nucleic acid strand P according to the present invention and a typical use example thereof.
- the example shown in FIG. 5 shows an example in which the detection nucleic acid strand P is used to detect the presence and amount of a nucleic acid strand T having a base sequence complementary to the nucleic acid strand portion N.
- the detection nucleic acid chain P shown in FIG. 1 is described as a representative example (the detection nucleic acid chain P in FIGS. 2 to 4 may also be! /).
- the nucleic acid strand P for detection is reduced or quenched by the action of the quencher substance Q before the predetermined enzyme treatment (described later) is performed. .
- T the nucleic acid strand portion N and the nucleic acid strand T form a double strand (complementary strand) (complementary strand formation procedure).
- the symbol W in FIG. 5 indicates a double-stranded (complementary strand) portion generated by the above-described complementary strand formation procedure! (Hereinafter the same).
- FIG. 6 is a diagram schematically showing how a cut is formed in AP site X by AP-endonuclease E.
- the double-stranded (complementary-stranded) portion W (see Fig. 5) and the double-stranded W and double-stranded W (see Fig. 6) shortened by AP-endonuclease E are: Because the chain length is different, t (mel
- the buffer solution temperature condition in the reaction field R is not more than the temperature t at which the nucleic acid strand P for detection before cleavage by AP-endonuclease E can proceed or maintain the complementary strand formation reaction.
- nucleic acid for detection after cleavage with AP-endonuclease E More than temperature t at which strand fragments P and P (see Fig. 6) cannot maintain or proceed with complementary strand formation reaction
- FIG. 7 shows that the nucleic acid strand fragments P and P for detection cleaved by AP-endonuclease E (see FIG. 6) were dissociated from the nucleic acid strand T under the above-mentioned appropriate temperature condition t to form a single strand.
- Reaction field R
- FIG. 9 shows that the nucleic acid chain P for detection (not cleaved) and the nucleic acid chain fragment Pl for detection (cleaved by AP-endonuclease E) by changing the temperature conditions of the reaction field R
- FIG. 3 is a reference diagram showing the state of P2 duplex formation or dissociation.
- FIG. 9 will be specifically explained. Under the condition of temperature t below the temperature, the nucleic acid strand for detection P
- the nucleic acid strand for detection P forms the complementary strand.
- the reaction can proceed and the formed complementary strand can be maintained.
- the detection nucleic acid chain fragments P and P shortened by AP-endase E are extracted from the nucleic acid chain.
- the detection nucleic acid strand P is also dissociated into single strands, and AP-
- Nucleic acid strand fragments P and P for detection shortened by denuclease E are also released from nucleic acid strand T.
- the present invention introduces an excessive amount of the detection nucleic acid chain P relative to the reaction field R relative to the nucleic acid chain T, thereby obtaining "the nucleic acid chain T and the detection nucleic acid chain P.
- Complementary strand formation between see Fig. 5) " ⁇ " Shortening of detection nucleic acid strand P with AP-endonuclease E (see Fig. 6) " ⁇ ” Shortened detection nucleic acid strand fragments P and P Dissociation from nucleic acid strand T and accompanying fluorescence amplification (Fig.
- FIG. 8 is a diagram showing a basic flow of this cycle reaction.
- the presence of the nucleic acid strand ⁇ ⁇ is shown from a very small amount (quenched by the Taentia substance Q). Since a strong fluorescent signal derived from the fluorescent substance F (which has been lost) can be obtained from the reaction field R, nucleic acid strands can be detected with high sensitivity.
- the setting of the temperature condition of the reaction field R is important that the complementary strand formation between the nucleic acid strand ⁇ and the detection nucleic acid strand ⁇ can proceed. Therefore, the condition of temperature t exceeding t as shown in Fig. 9 is not preferable.
- the temperature conditions suitable for the present invention are, as described above, the temperature at which the detection nucleic acid strand P before cleavage by AP-endonuclease E can proceed or maintain the complementary strand formation reaction. and the detection nucleic acid strand fragments P and P (see FIG. 6) after cleavage with AP-endonuclease E cannot maintain or proceed with the complementary strand formation reaction.
- the temperature becomes higher than the temperature t (ie, temperature ⁇ t ⁇ t) (see Fig. 9 again).
- the detection nucleic acid chain fragments P and P have different chain lengths.
- both the nucleic acid strand fragments for detection P and P are solved from the nucleic acid strand ⁇ b 1 2
- a feature of the present invention common to all of the application methods exemplified below is that a very small amount of substance is produced by the above "cycle reaction” involving an excessive amount of the detection nucleic acid strand P introduced into the reaction field. This means that it is possible to detect a small amount of reaction, interaction, and change in material structure with high sensitivity. Since this is a duplicate, I will omit the detailed explanation for each case.
- FIG. 10 is a schematic diagram showing a concept of a first applied method example using the detection nucleic acid strand P according to the present invention.
- This first applied method example is useful for verifying whether or not the target base sequence region exists in the base sequence of the nucleic acid strand Y. For example, heredity It can be confirmed whether or not a specific response nucleotide sequence for a specific transcription factor is present in the promoter region located upstream of the child.
- FIG. 11 is a schematic diagram showing the concept of a second applied method example using the nucleic acid strand for detection P according to the present invention.
- a substance for example, a protein
- a detection target exists on the surface S of a solid phase (for example, a substrate, a bead, or a carrier).
- a solid phase for example, a substrate, a bead, or a carrier.
- the substance M If the substance M is present in the sample solution, the nucleic acid strand Z forming the complementary strand and the detection nucleic acid strand P are bound to the substance M, so that the complementary strand
- AP-endonuclease E acts to cut the complementary strand (AP portion X of the nucleic acid strand portion N constituting it) into a single strand. For this reason, even if the content of the substance M is extremely small, a strong fluorescence signal can be obtained from the fluorescent substance F constituting the detection nucleic acid chain P. Thereby, the substance M can be detected with high sensitivity.
- FIG. 12 is a schematic diagram showing the concept of a third applied method example using the nucleic acid strand for detection P according to the present invention. According to this third applied method example, it is possible to detect the reaction and interaction of substance L with substance K immobilized on surface S of a solid phase (eg, substrate, beads, etc.).
- a solid phase eg, substrate, beads, etc.
- a sample solution containing a substance L for verifying the presence or absence of a reaction or interaction with the substance K with respect to the reaction field R on which the surface S on which the substance K is previously immobilized is exposed. Then, the reaction field R is washed with a solution.
- Substance L contains nucleic acid chain Z with a predetermined base sequence.
- the nucleic acid strand portion N is complementary to the base sequence of the nucleic acid strand Z.
- the detection nucleic acid strand P is introduced, and then double-strand-specific AP-endonuclease E is introduced into the reaction field R.
- AP-endonuclease E specific to double strand acts on the complementary strand, and a cut is made in the complementary strand (AP site X of the nucleic acid strand portion N constituting it). It becomes a main chain. For this reason, even if the amount of reaction or interaction between substance K and substance L is very small, the fluorescence signal can be obtained from the fluorescent substance F that forms the nucleic acid chain P for detection.
- FIG. 13 is a schematic diagram showing the concept of a fourth applied method example using the detection nucleic acid strand P according to the present invention.
- a low molecular weight compound that can be a drug candidate for example, a compound that can bind to a receptor and become an agonist or antagonist can be detected or screened with high sensitivity.
- a sample solution containing a candidate substance V that can bind to or interact with the substance U is introduced toward the substance U existing on the solid phase surface S such as a substrate, and then Wash the reaction field R with a solution.
- Substance V is labeled in advance with nucleic acid strand Z having a predetermined base sequence.
- nucleic acid chain portion N complementary to the base sequence of the nucleic acid chain Z is present.
- the detection nucleic acid strand P to be introduced is introduced, and then double-strand-specific AP-endonuclease E is introduced into the reaction field R.
- the candidate substance V binds to each other, the candidate substance V is labeled in the reaction field R! /, And the nucleic acid chain Z and the detection nucleic acid chain P (the nucleic acid chain part N) are To form complementary strands
- a double-strand-specific AP-endonuclease E acts on the complementary strand, and the complementary strand (AP site X of the nucleic acid strand portion N constituting it) is cut to become a single strand. Therefore, even if the binding amount between substance U and candidate substance V is very small, nucleic acid strand P for detection is constituted. A strong fluorescent signal from fluorescent substance F is obtained.
- FIG. 14 is a schematic diagram showing the concept of a fifth applied method example using the nucleic acid strand for detection P according to the present invention.
- environmental hormones endocrine disrupting substances
- transcription factor J DNA binding protein
- DNA binding protein changes its structure when hormone H binds to it, so that it binds to the response base sequence region existing in the upstream region of the gene, and It has a function of promoting transcription.
- endocrine disruptors called environmental hormones are substances that behave in the same way as hormone H in the body.
- a transcription factor (an intranuclear receptor) J is previously present (for example, immobilized) on a solid phase surface S such as a substrate or a bead, and a nucleic acid corresponding to the response base sequence region.
- Nucleic acid strand P for detection having nucleic acid strand portion N complementary to the base sequence of strand Z and nucleic acid strand Z
- a hormone-like substance h is introduced into the reaction field R of such a substance environment in order to verify whether it can become an endocrine disruptor.
- the hormone-like substance h has a function similar to that of hormone H, the structure of transcription factor J is changed by the action of hormone-like substance h.
- the reaction field R where the transcription factor J that has undergone this structural change is present, a nucleic acid chain Z corresponding to the above-mentioned response base sequence region and a nucleic acid chain portion N that forms a double strand with the nucleic acid chain Z
- a nucleic acid strand P for detection is further introduced, and a double strand-specific AP-endonuclease E is introduced into the reaction field R.
- a different AP-endonuclease E acts to break the complementary strand (AP site X of the nucleic acid strand portion N constituting it) into a single strand.
- FIG. 15 is a schematic diagram showing the concept of a sixth applied method example using the detection nucleic acid strand P according to the present invention. According to this sixth applied method example, for example, detection of a post-translational modification reaction of a protein can be performed.
- a protein may be modified after translation via an enzymatic reaction or the like.
- phosphorylation is a typical example.
- a protein D to be detected is previously present (for example, immobilized) on the solid phase surface S such as a substrate, and the protein D is not modified (for example, phosphorylated).
- Antibody B that specifically binds to antibody B and antibody B that specifically binds to the modified (eg, phosphorylated) portion should also be present.
- antibody B and antibody B have a nucleic acid chain consisting of a unique base sequence in advance.
- the detection nucleic acid strand Pa has a nucleic acid strand portion Na complementary to the nucleic acid strand Za, and the nucleic acid strand portion Nb complementary to the nucleic acid strand Zb.
- the detection nucleic acid strand Pb is introduced together.
- the detection nucleic acid chain Pa is labeled with a fluorescent substance F that can emit fluorescence of a predetermined wavelength
- the detection nucleic acid chain Pb is a fluorescent substance that can emit fluorescence with a wavelength different from that of the fluorescent substance F. Try to label F.
- the detection nucleic acid chain Pa binds to all the proteins D present in the reaction field R via the complementary chain, so that the fluorescent substance F constituting the detection nucleic acid chain Pa
- the fluorescent substance F constituting the detection nucleic acid chain Pa
- the modified nucleic acid D is bound to the detection nucleic acid strand Pb via a complementary strand.
- the number of molecules of the modified protein D can be predicted.
- FIG. 16 and 17 are schematic diagrams showing the concept of a seventh applied method example using the detection nucleic acid strand P according to the present invention.
- FIG. 16 is a schematic diagram when a substance (for example, protein) before structural change is detected
- FIG. 17 is a schematic diagram when a substance (for example, protein) after structural change is detected. According to this seventh applied method example, it is possible to detect the structural change of a substance and the numerical ratio of the structural change.
- a protein may exhibit a specific function by the action of other factors to change its higher-order structure. Therefore, the function and activity of the substance can be known by detecting such a structural change of the substance.
- a specific description will be given based on FIGS. 16 and 17.
- the symbol Dx shown in FIG. 16 indicates the protein before the structural change, while the symbol Dy shown in FIG. 17 indicates the protein after the structural change.
- an antibody B nucleic acid chain Za is labeled
- antibody B that specifically binds to the structural site dl of protein Dx to reaction field R where protein Dx immobilized on surface S is present.
- antibody B that specifically binds to the structural site d2 of the protein Dx (labeled with nucleic acid chain Zb), fluorescence wavelength
- the nucleic acid strand portion Na constituting the detection nucleic acid strand Pa is complementary to the nucleic acid strand Za labeled with the antibody B, and thus forms a double strand, constituting one detection nucleic acid strand Pb.
- Nucleic acid strand part Nb is complementary to the nucleic acid strand Zb labeled with antibody B, forming a double strand
- the reaction site R where the protein Dy immobilized on the surface S (a protein having a structural change) is present is characteristic of the structural site dl of the protein Dx (see FIG. 16).
- Acid chain Zb is labeled) and two types of nucleic acid chains Pa and Pb for detection with different fluorescence wavelengths are introduced.
- the protein Dy present on the surface S has undergone a modification that causes a structural change, and is not a protein Dx that has not undergone a structural change.
- both proteins Dx and Dy can be detected simultaneously by analyzing the fluorescence signal. For example, it can be applied to the detection of trace amounts of / 3 amyloid structural change, and thus diagnosis of Alzheimer's disease.
- FIG. 18 is a schematic diagram showing the concept of a seventh application example using the detection nucleic acid strand P according to the present invention.
- the seventh application example is an example in which the nucleic acid strand for detection P is used as a probe for a DNA chip.
- the nucleic acid strand P for detection according to the present invention can also be applied to other sensor technologies other than DNA chips.
- Complementary strands are successively formed between the nucleic acid strand portions N of P 1 and P 2.
- nucleic acid strand N When the nucleic acid strand N is fragmented (shortened) by a double strand-specific AP-endonuclease (not shown in FIG. 18), the nucleic acid strand T of the detection nucleic acid strand fragment that has been shortened A series of reactions (cycle reactions) occur in which the dissociation of force occurs and the accompanying fluorescence amplification occurs.
- nucleic acid strand for detection P when used as a probe for a DNA chip, a single nucleic acid strand T as a target is composed of a plurality of probes ( In the example shown in Fig. 18, it is used by P 1, P 2, P 2, and P), so highly sensitive detection is possible.
- the concentration of the target can be measured from the reaction rate.
- the force required to label the target nucleic acid strand T with a fluorescent dye can be saved. Furthermore, in the conventional DNA chip, the hybridized target nucleic acid strand T is fixed to the probe, so that the apparent concentration of the target nucleic acid strand T near the hybridization reaction field decreases, and the efficiency of the hybridization is reduced.
- the detection nucleic acid strand P according to the present invention is used, the target nucleic acid strand T is not fixed, and the target nucleic acid strand T in the vicinity of the hybridization reaction field is apparent. It can be expected that the concentration does not decrease and the efficiency of the hybridization is improved.
- nucleic acid chain portion SEQ ID NO: 2
- FIG. Etc a cycle reaction involving re-reference
- SEQ ID NO: 3 a large amount of nucleic acid chain fragments for detection were generated by the action of AP-endonuclease
- the AP-endonuclease used in this experiment is human AP-endonuclease (APE 1), and the fluorescent substance used for the nucleic acid strand for detection is the fluorescent dye FAM (bound to the 5 'end).
- FAM fluorescent dye
- TAMRA attachmented to the 3 'end.
- AP-endonuclease was added to the reaction field where lOOfmol target nucleic acid strand and 8 pmol of detection nucleic acid strand (having an AP site) existed for reaction.
- the reaction is performed at 37, 42, and 47 ° C within the appropriate temperature, and these reaction solutions are added with AP-endase!
- Electrophoresis was performed, separation was performed according to the size of the nucleic acid strand for detection, and detection was performed using a bound fluorescent dye.
- the degree V can be expressed by a linear equation. Therefore, it was shown that the target nucleic acid strand can be quantified in the same concentration range (see FIG. 21).
- This experimental example shows that an example of the application method according to the present invention was actually implemented and succeeded. Specifically, this experimental example detected the BMAL1-CLOCK complex, which is a biological clock gene product (clock protein) having transcription factor activity from human oral mucosal epithelial cells. It is an experimental example which shows that the detection of was successful.
- BMAL1-CLOCK complex which is a biological clock gene product (clock protein) having transcription factor activity from human oral mucosal epithelial cells. It is an experimental example which shows that the detection of was successful.
- the “nucleic acid strand for detection” used in this experimental example has the following base sequence structure in which a fluorescent dye (FITC) is bound to the 5 ′ end and a quencher substance (BHQ) is bound to the 3 ′ end.
- the oligo DNA provided is the sixth base from the 5 'end (missing guanine: AP site) (see Table 1).
- the nucleic acid strand that forms a double strand with this nucleic acid strand for detection is shown in Table 2.
- the anti-BMALl antibody (Santa Cruz Biotechnology) was used as the antibody.
- antibody magnetic beads were prepared by the following procedure. The above-described antibody was immobilized using magnetic beads (micromer-M [PE G-COOH], micromod PartiKeltechnologie ⁇ Boko PolyLink-Protein coupling Kit for COOOH Microparticles (Polyscience)).
- the antibody magnetic beads were set to 10 mM Tris-Cl (pH 7.5), 50 mM C1, 2.5 2.27926e + 2891ycerol, lOmM EDTA, 0 • 05P-40, 0.05 mg / mL salmon sperm DNA, 0.2 each.
- the suspension was suspended in a ⁇ M detection nucleic acid probe (double-stranded DNA comprising probe nucleic acids 1 and 2) and incubated at 37 ° C. lhr.
- the antibody magnetic beads were washed three times with 500 L of PBS-T to wash away nonspecifically bound molecules and probe nucleic acids.
- the so-called clock protein is a transcription factor and the most important circadian rhythm in biological rhythm. It is an autonomous oscillator that moves the music.
- a transcription factor, clock protein induces the expression of other clock proteins, and the induced clock protein also functions as a transcription factor and represses transcription of other (original) clock proteins.
- This negative feed loop is currently considered to function as a vibrator for a strong deer rhythm.
- the clock protein that is a transcription factor is a core of the biological clock, it is considered that measuring this clock protein is most suitable for measuring the biological rhythm of an organism.
- the human sleep-wake cycle is constrained by social life that is not limited to the autonomous control of the body clock, so there is a gap between the rhythm of the sleep-wake cycle and the autonomous rhythm of the body clock. It may cause poor physical condition as typified by jet lag and also the above-mentioned poor health condition.
- the amount of enzymes that metabolize drugs in the body has a strong dian rhythm, and the amount of molecules targeted by drugs often has a strong dian rhythm. It is known that there is a rhythm, and the idea of chronotherapy with a prescribed medication time is spreading.
- the present invention a predetermined experiment was performed to demonstrate that the expression variation of the BMALl-CLOCK protein complex can be observed from Hela cells.
- the promoter region has an E-box sequence to which the BMAL 1-CLOCK protein complex binds, and the fluctuation pattern of the mRNA, Perl, Per 2, which is activated by the BMALl-CLOCK protein complex, is observed. Comparison with expression change of BMALl-CLOCK protein complex.
- BMALl-CLOCK protein complex was detected in the same manner as oral mucosal epithelial cells (Fig. 24). (See drawing substitute graph).
- the primer sequences used in RT-PCR are shown in “Table 3” below.
- FIG. 25 is a diagram (drawing substitute graph) showing the results of quantifying the amount of Perl mRNA
- FIG. 26 is a diagram (drawing substitute graph) showing the results of quantifying the amount of Per2 mRNA.
- the mRNA levels of Perl and Per2 were calculated as values relative to the amount of GAPDH (glyceraldehy de-3-phosphate dehydrogenase) as an endogenous control.
- the fluctuation pattern of the amount of BMAL1-CLOCK protein complex is similar to the fluctuation pattern of Perl and Per2 mRNA whose transcription is activated by the BMAL1-CLOCK protein complex ( Figures 24 and 25). And comparison with FIG. 26), it was shown that the BMAL1-CLOCK protein complex quantity S reflects the transcriptional activity of the BMAL1-CL0CK protein complex. From these results, it was demonstrated that by applying the present invention, it is possible to detect clock proteins and to observe in detail the “expression fluctuation” of the BMAL1-CLOCK protein complex. .
- Some transcription factors have transcription activity only after binding to steroid hormones. There are many nuclear receptors. By applying the present invention, it is also possible to search and quantify these nuclear receptor ligands (steroid hormones, etc.). It is also possible to search, quantify, and evaluate drugs that act as agonists and antagonists for nuclear receptors. If SREBP activated by cholesterol is measured, metabolic syndrome and Alzheimer's can be prevented, and nutritional status can be determined by measuring transcription factors that serve as nuclear receptors for vitamin N honoremon. Ability to know physiological conditions including Using purified nuclear receptors, it is possible to quantify vitamins and hormones as ligands and to measure affinity with nuclear receptors, and screen for antagonists and antagonists that target nuclear receptors. It is also possible to apply the present invention to evaluation.
- the present invention can be used as a technique for detecting a very small amount of substance present in a reaction field with high sensitivity. For example,
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US12/377,560 US20100291553A1 (en) | 2006-08-14 | 2007-08-14 | Nucleic acid strand useful in detecting substance and method thereof |
CN2007800304414A CN101506363B (zh) | 2006-08-14 | 2007-08-14 | 用于检测物质的核酸链及其方法 |
EP07792487.6A EP2050817B1 (en) | 2006-08-14 | 2007-08-14 | Nucleic acid strand useful in detecting substance and method thereof |
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JP2007203079A JP5088034B2 (ja) | 2006-08-14 | 2007-08-03 | 物質の検出等に有用な核酸鎖とその方法 |
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WO2011087707A1 (en) * | 2009-12-22 | 2011-07-21 | Elitech Holding B.V. | Hypertheromostable endonuclease iv substrate probe |
JP5697129B2 (ja) * | 2010-03-24 | 2015-04-08 | 国立大学法人埼玉大学 | Fretを利用した酵素活性測定基質及びその製造方法 |
CN102296116A (zh) * | 2011-09-02 | 2011-12-28 | 北京大学 | 对dna目标序列进行信号放大和检测的方法 |
KR20140129221A (ko) * | 2012-05-21 | 2014-11-06 | 에스알씨, 인코퍼레이트 | 리신과 기타 리보솜 비활성화 단백질의 검출을 위한 방법과 시스템 |
TW201531569A (zh) * | 2014-02-12 | 2015-08-16 | Taiwan Sugar Corp | 雙股核酸分子與s1核酸酶於檢測核酸修復酵素活性的用途 |
CN103882128A (zh) * | 2014-03-13 | 2014-06-25 | 北京大学 | 常温下对目标dna序列进行信号放大和检测的方法 |
JP6640843B2 (ja) * | 2014-09-30 | 2020-02-05 | コーニンクレッカ フィリップス エヌ ヴェKoninklijke Philips N.V. | 第1及び第2のエピトープの空間的近接を検出する方法 |
WO2018008435A1 (ja) * | 2016-07-04 | 2018-01-11 | 株式会社ダナフォーム | 核酸分析方法 |
JP7270254B2 (ja) | 2017-06-16 | 2023-05-10 | デューク ユニバーシティ | 改善された標識検出、演算、検体感知、および調整可能な乱数生成のための共鳴体ネットワーク |
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- 2007-08-14 CN CN2007800304414A patent/CN101506363B/zh not_active Expired - Fee Related
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EP2050817A4 (en) | 2010-05-26 |
JP5088034B2 (ja) | 2012-12-05 |
US20100291553A1 (en) | 2010-11-18 |
JP2008067694A (ja) | 2008-03-27 |
EP2050817A1 (en) | 2009-04-22 |
EP2050817B1 (en) | 2013-05-01 |
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