WO2017098746A1 - Détecteur destiné à l'analyse du cortisol, procédé d'analyse du cortisol, réactif d'évaluation du stress, procédé d'évaluation du stress, réactif de test destiné à une maladie liée au cortisol, et procédé de test destiné au risque de contraction d'une maladie liée au cortisol - Google Patents

Détecteur destiné à l'analyse du cortisol, procédé d'analyse du cortisol, réactif d'évaluation du stress, procédé d'évaluation du stress, réactif de test destiné à une maladie liée au cortisol, et procédé de test destiné au risque de contraction d'une maladie liée au cortisol Download PDF

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WO2017098746A1
WO2017098746A1 PCT/JP2016/071937 JP2016071937W WO2017098746A1 WO 2017098746 A1 WO2017098746 A1 WO 2017098746A1 JP 2016071937 W JP2016071937 W JP 2016071937W WO 2017098746 A1 WO2017098746 A1 WO 2017098746A1
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region
cortisol
sensor
nucleic acid
polynucleotide
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PCT/JP2016/071937
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Japanese (ja)
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金子　直人
宏貴 皆川
穣 秋冨
克紀 堀井
行大 白鳥
巌 和賀
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Ｎｅｃソリューションイノベータ株式会社
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Priority to JP2017554933A priority Critical patent/JP6642859B2/ja
Publication of WO2017098746A1 publication Critical patent/WO2017098746A1/fr

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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
    • 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
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a sensor for cortisol analysis, a cortisol analysis method, a stress evaluation reagent, a stress evaluation method, a test reagent for cortisol-related diseases, and a method for testing the possibility of cortisol-related diseases.
  • Non-Patent Document 1 It has been clarified that cortisol concentration in body fluids such as blood, urine and saliva correlates with stress. For this reason, it has been attempted to measure the degree of stress by measuring the concentration of cortisol in saliva that can be easily collected.
  • an object of the present invention is to provide a new sensor for cortisol analysis, a cortisol analysis method, a stress evaluation reagent, a stress evaluation method, a cortisol-related disease test reagent, and a cortisol-related disease morbidity possibility that can be used for cortisol analysis. It is to provide a method of testing.
  • the sensor for analyzing cortisol of the present invention includes the following (I) including a binding region (A) that binds to a target and a G-forming region (D) that forms a G-quartet structure.
  • a binding region (A) that binds to a target and a G-forming region (D) that forms a G-quartet structure.
  • the target is cortisol
  • the G-forming region (D) becomes inactive due to inhibition of formation of the G-quartet structure
  • the G-forming region (D) is activated by forming a G-quartet structure.
  • the binding region (A) has a stem formation region (S D ), an intermediate region (C), and a stem formation region (S C ),
  • the stem formation region (S D ) has a sequence complementary to the G formation region (D)
  • the stem-forming region (S C ) is a single-stranded nucleic acid molecule having a sequence complementary to the intermediate region (C).
  • the G formation region (D) includes a first region (D1) and a second region (D2), and a region in which a G-quartet is formed by the first region (D1) and the second region (D2) And A single-stranded nucleic acid molecule having the first region (D1) on one end side of the binding region (A) and the second region (D2) on the other end side of the binding region (A).
  • (III) a double-stranded nucleic acid molecule composed of a first strand (ss1) and a second strand (ss2),
  • the first strand (ss1) has the G-forming region (D) and the binding region (A) in this order
  • the second chain (ss2) has a stem formation region (S D ) and a stem formation region (S A ) in this order, and the stem formation region (S D ) is in the G formation region (D).
  • the cortisol analysis method of the present invention includes a contact step of bringing a sample into contact with the sensor for cortisol analysis of the present invention, and binding between cortisol in the sample and the sensor for cortisol analysis. It comprises a detection step of detecting cortisol in the sample by combining with the region (A).
  • the stress evaluation reagent of the present invention includes the aforementioned sensor for cortisol analysis of the present invention.
  • the stress evaluation method of the present invention (hereinafter also referred to as “evaluation method”) is a contact step of bringing a sample of a subject into contact with the sensor for cortisol analysis of the present invention, A detection step of detecting cortisol in the sample by binding cortisol in the sample and the binding region (A) in the sensor for cortisol analysis, and comparing the amount of cortisol in the detection step with a reference value Thus, an acquisition step for acquiring information on stress is included.
  • test reagent includes the sensor for cortisol analysis of the present invention.
  • a method for testing the possibility of cortisol-related disease of the present invention comprises a contact step of contacting a sample of a subject with the sensor for cortisol analysis of the present invention, A detection step for detecting cortisol in the sample by binding cortisol in the sample and a binding region (A) in the sensor for analyzing cortisol, and comparing the amount of cortisol in the detection step with a reference value
  • test method comprises a contact step of contacting a sample of a subject with the sensor for cortisol analysis of the present invention, A detection step for detecting cortisol in the sample by binding cortisol in the sample and a binding region (A) in the sensor for analyzing cortisol, and comparing the amount of cortisol in the detection step with a reference value
  • test method comprises a contact step of contacting a sample of a subject with the sensor for cortisol analysis of the present invention, A detection step for detecting cortisol in the sample by binding cortisol in the
  • the G-forming region (D) forms a G-quartet structure by cortisol binding to the binding region (A).
  • the G-forming region (D) in which the G-quartet structure is formed is an active type, and for example, it generates a catalytic function or emits fluorescence by forming a complex with porphyrin. For this reason, for example, by detecting the catalytic function or the fluorescence, cortisol can be easily analyzed.
  • the senor for analyzing cortisol according to the present invention has low cross-reactivity to similar compounds such as melatonin, L-tryptophan, cortisone, norepinephrine, epinephrine, and cholic acid. For this reason, the sensor for cortisol analysis of the present invention can specifically analyze cortisol, for example.
  • FIG. 1 is a graph showing the fluorescence intensity in a reaction solution using the analytical sensor of the present invention in Example 2.
  • FIG. 2 is a graph showing the absorbance in a reaction solution using the analytical sensor of the present invention in Example 3.
  • FIG. 3 is a graph showing the fluorescence intensity at each migration distance of the electrophoresis gel when the reaction solution using the analytical sensor of the present invention was electrophoresed in Example 4.
  • FIG. 4 is a graph showing the relative value of absorbance in a reaction solution using the analytical sensor of the present invention in Example 5.
  • FIG. 5 is a graph showing the degree of fluorescence polarization in a reaction solution using the analytical sensor of the present invention in Example 6.
  • the sensor for analyzing cortisol of the present invention includes the following (I), (II), and (II) including a binding region (A) that binds to a target and a G-forming region (D) that forms a G-quartet structure. III) comprising at least one nucleic acid molecule selected from the group consisting of The target is cortisol, In the absence of the target, the G-forming region (D) becomes inactive due to inhibition of formation of the G-quartet structure, In the presence of the target, the G-forming region (D) is activated by forming a G-quartet structure.
  • the binding region (A) has a stem formation region (S D ), an intermediate region (C), and a stem formation region (S C ),
  • the stem formation region (S D ) has a sequence complementary to the G formation region (D)
  • the stem-forming region (S C ) is a single-stranded nucleic acid molecule having a sequence complementary to the intermediate region (C).
  • the G formation region (D) includes a first region (D1) and a second region (D2), and a region in which a G-quartet is formed by the first region (D1) and the second region (D2) And A single-stranded nucleic acid molecule having the first region (D1) on one end side of the binding region (A) and the second region (D2) on the other end side of the binding region (A).
  • (III) a double-stranded nucleic acid molecule composed of a first strand (ss1) and a second strand (ss2),
  • the first strand (ss1) has the G-forming region (D) and the binding region (A) in this order
  • the second chain (ss2) has a stem formation region (S D ) and a stem formation region (S A ) in this order, and the stem formation region (S D ) is in the G formation region (D).
  • the region is also referred to as a nucleic acid region and cortisol as a target.
  • the single-stranded nucleic acid molecule and the double-stranded nucleic acid molecule in the present invention can also be referred to as, for example, a single-stranded nucleic acid element and a double-stranded nucleic acid element, respectively.
  • the inhibition of the formation of the G-quartet structure indicates that the switch-OFF (or turn-OFF) and the formation of the G-quartet structure indicate that the switch-ON (or (turn-ON).
  • the phrase “the other sequence is complementary to a certain sequence” means, for example, a sequence that can be annealed between the two. The annealing is also referred to as stem formation.
  • the term “complementary” means, for example, that complementarity when two kinds of sequences are aligned is, for example, 90% or more, preferably 95% or more, 96% or more, 97% or more, 98% or more. More preferably 99% or more, particularly preferably 100%, that is, completely complementary.
  • another sequence is complementary to a certain sequence when the sequence is directed from the 5 ′ side to the 3 ′ side, and the sequence is directed from the other 3 ′ side to the 5 ′ side. Means that the bases of each other are complementary.
  • the binding region (A) is a nucleic acid that binds to cortisol and is also referred to as a cortisol-binding nucleic acid.
  • the dissociation constant for cortisol in the binding region (A) is not particularly limited.
  • the binding between the binding region (A) and the cortisol can be determined by, for example, surface plasmon resonance molecular interaction (SPR) analysis.
  • SPR surface plasmon resonance molecular interaction
  • ProteON BioRad
  • BioRad BioRad
  • the cortisol is represented by the following formula (1).
  • the cortisol may be a derivative such as an isomer, a salt, a hydrate, or a solvate.
  • the description regarding the said cortisol can be used for the said derivative
  • the binding region (A) includes, for example, at least one polynucleotide selected from the group consisting of L (a1) to (a3) and (a4).
  • A1 Poly consisting of a base sequence enclosed in a square in any one of the base sequences of SEQ ID NOs: 1 to 279 of Tables 1A to H, SEQ ID NOs: 280 to 299 of Table 2, and SEQ ID NOs: 300 to 427 of Tables 3A to D
  • Nucleotide (a2) A polynucleotide (a3) consisting of a base sequence in which one or several bases are deleted, substituted, inserted and / or added in any of the base sequences of (a1), and which binds to the cortisol
  • the polynucleotide of (a1) is a square in any one of the nucleotide sequences of SEQ ID NOS: 1 to 279 in Tables 1A to H, SEQ ID NOs: 280 to 299 in Table 2, and SEQ ID NOs: 300 to 427 in Tables 3A to D. It is a polynucleotide consisting of an enclosed base sequence.
  • “one or several” may be in the range where the polynucleotide (a2) binds to cortisol, for example.
  • the “one or several” is, for example, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 or 2, 1 in the base sequence of any of (a1). It is.
  • the numerical range of numbers such as the number of bases and the number of sequences, for example, discloses all positive integers belonging to the range. That is, for example, the description “1 to 5 bases” means all disclosures of “1, 2, 3, 4, 5 bases” (the same applies hereinafter).
  • the “identity” may be, for example, within a range in which the polynucleotide (a3) binds to cortisol.
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the identity can be calculated with default parameters using analysis software such as BLAST and FASTA (hereinafter the same).
  • the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide (a1).
  • the hybridization can be detected by, for example, various hybridization assays.
  • the hybridization assay is not particularly limited, for example, Zanburuku (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) ,” [Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.
  • the “stringent conditions” may be, for example, any of low stringent conditions, medium stringent conditions, and high stringent conditions.
  • Low stringent conditions are, for example, conditions of 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C.
  • Medium stringent conditions are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 42 ° C.
  • “High stringent conditions” are, for example, conditions of 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C.
  • the degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, time, and the like.
  • “Stringent conditions” are, for example, Zanburuku previously described (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) ,” [Cold Spring Harbor Laboratory Press ( 1989)] etc. may be employed.
  • the binding region (A) may be, for example, a molecule composed of any one of the polynucleotides (a1) to (a3) and (a4) or a molecule containing the polynucleotide.
  • the binding region (A) may further have, for example, a linker sequence and / or an additional sequence in addition to the polynucleotide.
  • the length of the binding region (A) is not particularly limited, and the lower limit is, for example, 12 base length, 15 base length, 18 base length, and the upper limit is, for example, 140 base length, 80 base length and 60 base length, and the range is, for example, 12 to 140 base length, 15 to 80 base length, and 18 to 60 base length.
  • the polynucleotide of (a1) is a polynucleotide comprising a base sequence surrounded by a square in any of the base sequences of SEQ ID NOs: 1 to 279
  • the polynucleotides of (a2) to (a4) are, for example, In the base sequences of the respective sequence numbers in Table 6 below, it is preferable that the corresponding conserved base sequences are conserved.
  • the conserved base sequence is aligned with reference to the base sequence of each SEQ ID NO of the polynucleotide (a1), for example, and the corresponding (matching) base sequence is stored. It can be judged that the nucleotide sequence is.
  • the identity is, for example, 50% or more, 60% or more, 70% or more, 80 %, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.
  • the G formation region (D) forms a G quartet structure
  • a catalytic function and fluorescence occur.
  • the sensor of the present invention for example, in the absence of the target, formation of the G quartet structure of the G formation region (D) is inhibited, and in the presence of the target, the binding between the target and the binding region (A) A G quartet structure of the G formation region (D) is formed.
  • the sensor of the present invention can detect the target by, for example, detecting at least one of the catalytic function and the generation of fluorescence due to the formation of the G quartet structure.
  • the G-forming region (D) when the G-forming region (D) forms a G-quartet structure, for example, it exhibits the two types of properties (catalytic function or fluorescence).
  • the sensor of the present invention can be used.
  • the G-forming region (D) is only required to form a G-quartet structure, and the catalytic function and the presence or absence of fluorescence are not particularly limited.
  • the G-forming region (D) forms the G-quartet structure
  • a complex with porphyrin is formed, whereby the complex emits fluorescence.
  • the G-quartet nucleic acid is also called a fluorescent nucleic acid.
  • the sensor of the present invention inhibits formation of a G-quartet structure in the G-forming region (D) in the absence of a target. Therefore, no fluorescence is emitted from the complex, and the switch is turned off. On the other hand, in the presence of the target, a G-quartet structure is formed in the G-forming region (D). In order to form a complex with porphyrin, fluorescence from the complex is emitted, and the switch is turned on. For this reason, for example, the presence or absence of the target or the target amount can be analyzed by fluorescence due to the complex formation between the G-forming region (D) and the porphyrin.
  • the G-forming region (D) forms the G-quartet structure
  • a complex with porphyrin is formed, thereby causing the catalytic function of the enzyme.
  • the G-quartet nucleic acid is also called a catalytic nucleic acid.
  • the catalytic function is, for example, a catalytic function of a redox reaction.
  • the oxidation-reduction reaction is, for example, a reaction that causes transfer of electrons between two substrates in the process of generating a product from the substrates.
  • the kind of the redox reaction is not particularly limited.
  • the catalytic function of the oxidation-reduction reaction includes, for example, the same activity as an enzyme, and specifically includes, for example, the same activity as peroxidase (hereinafter referred to as “peroxidase-like activity”).
  • peroxidase activity examples include horseradish peroxidase (HRP) activity.
  • the G-forming region (D) is generally called a DNA enzyme or DNAzyme in the case of a DNA sequence, and is called an RNA enzyme or RNAzyme in the case of an RNA sequence.
  • DNAzyme examples include nucleic acid molecules such as the following articles (1) to (4). (1) Travascio et al., Chem. Biol., 1998, vol.5, p.505-517 (2) Cheng et al., Biochemistry, 2009, vol.48, p.7817-7823 (3) Teller et al., Anal. Chem., 2009, vol.81, p.9114-9119 (4) Tao et al., Anal. Chem., 2009, vol.81, p.2144-2149
  • the sensor of the present invention inhibits formation of a G-quartet structure in the G-forming region (D) in the absence of a target. No complex is formed, and therefore the catalytic function of the complex does not occur and the switch is turned OFF. On the other hand, in the presence of the target, a G-quartet structure is formed in the G-forming region (D). In order to form the composite, the catalytic function of the composite is generated and the switch is turned on. Therefore, for example, by using a substrate corresponding to the catalytic function together, the presence or absence of the target or the target amount can be analyzed by the catalytic function due to the complex formation between the G-forming region (D) and the porphyrin.
  • the analytical sensor of the present invention can detect, for example, fluorescence by the complex without changing the sequence of the G-forming region (D), and in the presence of the substrate,
  • the catalytic function can also be detected by the complex.
  • the porphyrin that forms a complex with the G-forming region (D) is not particularly limited, and examples thereof include unsubstituted porphyrin and derivatives thereof.
  • the derivatives include substituted porphyrins, metal porphyrins formed with complexes with metal elements, and the like.
  • the substituted porphyrin include N-methylmesoporphyrin (NMM), Zn-DIGP, ZnPP9, and TMPyP.
  • Examples of the metal porphyrin include hemin, which is a trivalent iron complex.
  • the porphyrin when the catalytic function is caused, is preferably, for example, the metal porphyrin, and more preferably hemin.
  • the porphyrin when generating the fluorescence in the G-forming region (D), is preferably NMM, Zn-DIGP, ZnPP9, TMPyP, or the like.
  • the G-forming region (D) may be, for example, a single-stranded type or a double-stranded type.
  • the single-stranded type can form, for example, a G-quartet structure in a single-stranded G-forming region (D).
  • the double-stranded type includes, for example, a first region (D1) and a second region (D2), and a G-quartet structure is formed between the first region (D1) and the second region (D2). Can be formed.
  • the latter double-stranded type includes, for example, a structure in which the first region and the second region are indirectly linked, and will be specifically described in the nucleic acid molecule (II) described later.
  • the G-forming region (D) is, for example, at least one polynucleotide selected from the group consisting of the following (d1) to (d3) and (d4) including.
  • (D1) a polynucleotide comprising the underlined base sequence in any of the base sequences of SEQ ID NOs: 1 to 279 of Tables 1A to H and SEQ ID NOs: 300 to 427 of Tables 3A to D
  • (d2) any of the above (d1) A polynucleotide comprising the base sequence in which one or several bases are deleted, substituted, inserted and / or added, and forming the G-quartet structure
  • (d3) any one of (d1) A polynucleotide comprising the base sequence having 80% or more identity to the base sequence and forming the G-quartet structure
  • a polynucleotide comprising any one of the base sequences of (d1) A complementary nucleotide sequence to a polynucleotide that hybridizes under stringent conditions to form the G-quartet structure.
  • the polynucleotide (d1) is a polynucleotide comprising an underlined base sequence in any one of the base sequences of SEQ ID NOs: 1 to 279 of Tables 1A to H and SEQ ID NOs: 300 to 427 of Tables 3A to D. .
  • “one or several” may be in the range where the polynucleotide of (d2) forms the G-quartet structure, for example.
  • the “one or several” is, for example, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1, or 2 in any of the base sequences of (d1).
  • identity may be, for example, within a range in which the polynucleotide of (d3) forms the G-quartet structure.
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (d1).
  • “hybridization” and “stringent conditions” the above description can be used.
  • the G-forming region (D) may be, for example, a molecule composed of any of the polynucleotides (d1) to (d4) or a molecule containing the polynucleotide. In the latter case, the G-forming region (D) may further have, for example, a linker sequence and / or an additional sequence in addition to the polynucleotide.
  • the length of the single-stranded G-forming region (D) is not particularly limited, and the lower limit is, for example, 11 base length, 13 base length, 15 base length, and the upper limit is, for example, 60 base length, It is 36 bases long and 18 bases long.
  • one of the first region (D1) and the second region (D2) is, for example, a group consisting of the following (e1) to (e3) and (e4)
  • the other region includes at least one polynucleotide selected from the group consisting of (f1) to (f3) and (f4) below.
  • the following polynucleotides (e2) to (e4) are, for example, polynucleotides that form a G-quartet structure with at least one of the following polynucleotides (f1) to (f3) and (f4).
  • polynucleotides (f2) to (f4) are, for example, polynucleotides that form a G-quartet structure with at least one of the following polynucleotides (e1) to (e3) and (e4).
  • (E1) a polynucleotide comprising the underlined base sequence in any one of SEQ ID NOS: 280 to 299 in Table 2 (e2) In the base sequence of any one of (e1), one or several bases Polynucleotide comprising a nucleotide sequence deleted, substituted, inserted and / or added (e3) Polynucleotide comprising a nucleotide sequence having 80% or more identity to any of the nucleotide sequences of (e1) (E4) a polynucleotide comprising a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to the polynucleotide comprising any one of the base sequences of (e1)
  • (F1) a polynucleotide comprising the underlined base sequence enclosed in parentheses in the base sequence of SEQ ID NO: (e1)
  • (f2) In the base sequence of any one of (f1), one or several bases are Polynucleotide consisting of a base sequence deleted, substituted, inserted and / or added (f3)
  • (F4) a polynucleotide comprising a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to the polynucleotide comprising any one of the base sequences (f1)
  • the polynucleotide (e1) is a polynucleotide comprising the underlined base sequence in any one of the sequence numbers 280 to 299 in Table 2.
  • “1 or several” is, for example, within a range in which the polynucleotide of (e2) forms a G-quartet structure with at least one of the polynucleotides (f1) to (f4). That's fine.
  • the “one or several” is, for example, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1, or 2 in any one of the base sequences of (e1).
  • the “identity” is, for example, within the range in which the polynucleotide of (e3) forms the G-quartet structure with at least one of the polynucleotides (f1) to (f4). Good.
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (e1).
  • “hybridization” and “stringent conditions” the above description can be used.
  • the polynucleotide (f1) is a polynucleotide comprising an underlined base sequence surrounded by parentheses in any one of the base sequences of SEQ ID NOs: 280 to 299 in Table 2.
  • “1 or several” is, for example, within a range in which the polynucleotide of (f2) forms a G-quartet structure with at least one of the polynucleotides (e1) to (e4). That's fine.
  • the “one or several” is, for example, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1, or 2 in any of the base sequences of (f1).
  • the “identity” is, for example, within a range in which the polynucleotide of (f3) forms the G-quartet structure with at least one polynucleotide of (e1) to (e4). Good.
  • the “identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (f1).
  • “hybridization” and “stringent conditions” the above description can be used.
  • the length of the first region (D1) and the second region (D2) is not particularly limited, and both may be the same or different.
  • the length of the first region (D1) is not particularly limited, and the lower limit is, for example, 7 base length, 8 base length, 10 base length, and the upper limit is, for example, 30 base length, 20 base length, 10
  • the base length and the range thereof are, for example, 7 to 30 base length, 7 to 20 base length, and 7 to 10 base length.
  • the length of the second region (D2) is not particularly limited, and the lower limit is, for example, 7 base length, 8 base length, 10 base length, and the upper limit is, for example, 30 base length, 20 base length, 10
  • the base length and the range thereof are, for example, 7 to 30 base length, 7 to 20 base length, and 7 to 10 base length.
  • each of (I), (II), and (III) will be described below.
  • description of each nucleic acid molecule can each be used.
  • nucleic acid molecule (I) The nucleic acid molecule (I) has the G-forming region (D) and the binding region (A), The binding region (A) has a stem formation region (S D ), an intermediate region (C), and a stem formation region (S C ), The stem formation region (S D ) has a sequence complementary to the G formation region (D), The stem forming region (S C ) is a single-stranded nucleic acid molecule having a sequence complementary to the intermediate region (C).
  • the G-forming region (D) is, for example, the single-stranded type.
  • the G-quartet formation of the G-forming region (D) is controlled to be ON-OFF depending on the presence or absence of a target.
  • the present invention is not limited to this mechanism.
  • the nucleic acid molecule (I) is annealed between the G-forming region (D) and the stem-forming region (S D ) in the molecule, so that the G-forming region (D) Formation of the G-quartet structure is inhibited (switch-OFF), and as a result, for example, formation of a complex between the G-forming region (D) and porphyrin is inhibited.
  • the structure of the intermediate region (C) is also fixed by annealing the intermediate region (C) and the stem forming region (S C ) in the molecule.
  • the structure of the molecule in this state is also called an inactive type.
  • the nucleic acid molecule (I) is released from the annealing of the intermediate region (C) and the stem formation region (S C ) by the contact of the target with the binding region (A).
  • the three-dimensional structure of the binding region (A) changes to a more stable structure.
  • the annealing of the G formation region (D) and the stem formation region (S D ) is canceled, and a G-quartet structure is formed in the region of the G formation region (D) (switch-ON).
  • a complex of the G-forming region (D) and porphyrin is formed, and for example, a catalytic function and fluorescence are generated.
  • the structure of the molecule in this state is also called an active form. Therefore, for example, the nucleic acid molecule (I) does not cause the catalytic function and fluorescence due to the complex formation in the absence of the target, and the catalytic function and fluorescence due to the complex formation only in the presence of the target. Therefore, target analysis such as qualitative or quantitative is possible.
  • the stem formation region (S D ) preferably has a sequence that is entirely or partially complementary to a part of the G formation region (D).
  • the stem formation region (S C ) is, for example, a sequence that is entirely or partially complementary to a part of the intermediate region (C).
  • the order of the regions is such that the G-forming region (D) and the stem-forming region (S D ) are annealed in the molecule, and the intermediate region
  • the order of annealing of the region (C) and the stem formation region (S C ) may be sufficient.
  • the following order can be illustrated as a specific example. (1) 5'- D-S C -C-S D -3 ' (2) 5'- S D -C- S C -D -3 '
  • the formation of the G-quartet structure is turned on and off as follows.
  • the intermediate region (C) and the stem formation region (S C ), the G formation region (D) and the stem formation region (S D ) form a stem, and the G formation region ( Inhibits the formation of the G-quartet structure of D).
  • the respective stem formation is released by the contact of the target with the binding region (A), and a G-quartet structure is formed in the G formation region (D).
  • the stem formation region (S D ) is complementary to the 5′-side region of the G formation region (D), and the stem formation region (S C ) It is preferably complementary to the 5 ′ region of the region (C).
  • the regions may be connected directly or indirectly.
  • the direct connection means that, for example, the 3 ′ end of one region and the 5 ′ end of the other region are directly bonded, and the indirect connection is, for example, 3 of one region. It means that the “terminal” and the 5 ′ terminal of the other region are bonded via the intervening linker region (also referred to as “internal region”).
  • the intervening linker region may be, for example, a nucleic acid sequence or a non-nucleic acid sequence, preferably the former.
  • the nucleic acid molecule (I) preferably has, for example, two intervening linker regions that are non-complementary to each other as the intervening linker region.
  • the positions of the two intervening linker regions are not particularly limited.
  • the intervening linker region linked to the intermediate region (C) is (L 1 ) (also referred to as “internal region (I c )”), and the intervening linker region linked to the G-forming region (D) is ( L 2 ) (also referred to as “internal region (I D )”).
  • the nucleic acid molecule (I) may have, for example, both (L 1 ) and (L 2 ) as an intervening linker region, or may have only one of them.
  • the formation of the G-quartet structure is turned on and off as follows.
  • the intermediate region (C) and the stem forming region (S C ) the G forming region (D) and the stem forming region (S D ) form stems, respectively.
  • the intervening linker region (L 1 ) and the intervening linker region (L 2 ) form an internal loop that inhibits the formation of the G-quartet structure of the G-forming region (D).
  • the respective stem formation is released by the contact of the target with the binding region (A), and a G-quartet structure is formed in the G formation region (D).
  • the lengths of the stem-forming sequence (S C ) and the stem-forming sequence (S D ) are not particularly limited.
  • the length of the stem-forming sequence (S C ) is, for example, 1 to 60 bases long, 1 to 10 bases long, or 1 to 7 bases long.
  • the stem-forming sequence (S D ) has a length of, for example, 1 to 30 bases, 0 to 10 bases, 1 to 10 bases, 0 to 7 bases, or 1 to 7 bases.
  • the stem forming sequence (S C ) and the stem forming sequence (S D ) may have the same length, the former may be long, or the latter may be long.
  • the lengths of the intervening linker regions (L 1 ) and (L 2 ) are not particularly limited.
  • the lengths of the intervening linker regions (L 1 ) and (L 2 ) are, for example, 0 to 30 bases, 1 to 30 bases, 1 to 15 bases, and 1 to 6 bases, respectively.
  • the lengths of the intervening linker regions (L 1 ) and (L 2 ) may be the same or different, for example. In the latter case, the difference in length of the intervening linker region (L 1) and (L 2) is not particularly limited, for example, 1 to 10 bases in length, 1 or 2 bases in length, one base in length.
  • the binding region (A) is, for example, at least one polynucleotide selected from the group consisting of (a1) to (a4), wherein the polynucleotide of (a1) is It is a polynucleotide that has been replaced with a polynucleotide having a base sequence surrounded by a square in any of the base sequences of SEQ ID NOS: 1 to 279 in Tables 1A to H. The description can be used after the above replacement.
  • the G-forming region (D) is, for example, at least one polynucleotide selected from the group consisting of (d1) to (d4), and the polynucleotide of (d1), It is a polynucleotide that has been replaced with a polynucleotide comprising the underlined base sequence in any one of SEQ ID NOS: 1 to 279 in Tables 1A to H. The description can be used after the above replacement.
  • the nucleic acid molecule (I) is, for example, at least one polynucleotide selected from the group consisting of the following (s1) to (s3) and (s4).
  • (S1) A polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 279 (s2) In the nucleotide sequence of any of the above (s1), one or several bases are deleted, substituted, inserted and / or added
  • (S4) A polynucleotide having a function equivalent to that of (s1) (s4) From a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the base sequences of (s1) And a polynucleotide having a function equivalent to that of (s1)
  • “having the same function as (s1)” means that in the absence of the target, the G formation region (D) does not form a G-quartet structure, and in the presence of the target. This means that the target is bonded to the bonding region (A) and the G-forming region (D) forms a G-quartet structure.
  • the polynucleotide (s1) is a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 279.
  • one or several means, for example, 1 to 10, 1 to 7, 1 to 5, 1 to 5 in any of the base sequences of (s1). Three, one or two.
  • identity is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide (s1).
  • “hybridization” and “stringent conditions” the above description can be used.
  • the conserved base sequence is, for example, aligned based on the base sequence of each SEQ ID NO of the polynucleotide of (s1), and the corresponding base sequence is the conserved base It can be judged as an array.
  • the identity is, for example, 50% or more, 60% or more, 70% or more, 80% or more 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the length of the nucleic acid molecule (I) is not particularly limited.
  • the length of the nucleic acid molecule (I) is, for example, 40 to 120 bases long, 45 to 100 bases long, 50 to 80 bases long.
  • the nucleic acid molecule (II) has the G-forming region (D) and the binding region (A),
  • the G formation region (D) includes a first region (D1) and a second region (D2), and a region in which a G-quartet is formed by the first region (D1) and the second region (D2)
  • a single-stranded nucleic acid molecule having the first region (D1) on one end side of the binding region (A) and the second region (D2) on the other end side of the binding region (A). is there.
  • the G-forming region (D) is, for example, the double-stranded type (hereinafter also referred to as “split type”).
  • the split-type G-forming region (D) is a molecule that includes the first region (D1) and the second region (D2), and forms a G-quartet structure.
  • the first region (D1) and the second region (D2) may be any sequence that forms the G-quartet structure, and more preferably, a guanine quadruplex structure. It is the arrangement
  • the G-quartet formation of the G-forming region (D) is controlled to ON-OFF depending on the presence or absence of a target. Note that the present invention is not limited to this mechanism.
  • the first region (D1) and the second region (D2) that form a G-quartet structure in a pair form the binding region (A). Are spaced apart from each other.
  • the first region (D1) and the second region (D2) are arranged at a distance, in the absence of the target, the first region (D1) and the second region ( D2) is inhibited from forming a G-quartet structure (switch-OFF), and as a result, for example, complex formation between the G-forming region (D) and porphyrin is inhibited.
  • the structure of the molecule in this state is also called an inactive type.
  • the nucleic acid molecule (II) has a more stable structure in which the three-dimensional structure of the binding region (A) has a stem-loop structure by the contact of the target with the binding region (A). Change.
  • the first region (D1) and the second region (D2) approach each other with the change in the three-dimensional structure of the binding region (A), and the first region (D1) and the second region (D2).
  • a G-quartet structure is formed (switch-ON) between them and, as a result, for example, a complex of the G-forming region (D) and porphyrin is formed, and a catalytic function and fluorescence are generated.
  • the structure of the molecule in this state is also called an active form. Therefore, for example, the nucleic acid molecule (II) does not cause the catalytic function and fluorescence due to the complex formation in the absence of the target, and the catalytic function and fluorescence due to the complex formation only in the presence of the target. As a result, target analysis such as qualitative or quantitative is possible.
  • the nucleic acid molecule (II) uses a double-stranded type as the G-forming region (D), and the first region (D1) and the second region via the binding region (A). Region (D2) is arranged. For this reason, for example, it is not necessary to set conditions for each type of binding nucleic acid molecule that binds to cortisol, and a desired cortisol-binding nucleic acid molecule can be set as the binding region (A).
  • the first region (D1) and the second region (D2) may be arranged via the binding region (A), and any of the binding regions (A) You may arrange
  • the first region (D1) is disposed on the 5 ′ side of the coupling region (A)
  • the second region (D2) is disposed on the 3 ′ side of the coupling region (A). An example is shown.
  • the first region (D1) and the binding region (A) may be connected directly or indirectly, or the second region (D2) and The binding region (A) may be connected directly or indirectly.
  • the direct connection means that, for example, the 3 ′ end of one region and the 5 ′ end of the other region are directly bonded, and the indirect connection is, for example, 3 of one region.
  • Means that the 'terminal and the 5' end of the other region are linked via the intervening linker region; specifically, the 3 'end of one region and the 5' end of the intervening linker region Means that the 3 ′ end of the intervening linker region and the 5 ′ end of the other region are directly bound.
  • the intervening linker region may be, for example, a nucleic acid sequence or a non-nucleic acid sequence, preferably the former.
  • the nucleic acid molecule (II) has the intervening linker region (first linker region (L 1 )) between the first region (D1) and the binding region (A), It is preferable to have the intervening linker region (second linker region (L 2 )) between the second region (D2) and the binding region (A).
  • the first linker region (L 1 ) and the second linker region (L 2 ) may be either one or preferably both. When both the first linker region (L 1 ) and the second linker region (L 2 ) are included, the respective lengths may be the same or different.
  • the length of the linker region is not particularly limited, and the lower limit is, for example, 1, 3, 5, 7, 9 bases, and the upper limit is, for example, 20, 15, 10 bases.
  • the base sequence from the 5 ′ end side of the first linker region (L 1 ) and the base sequence from the 3 ′ end side of the second linker region (L 2 ) are non-complementary to each other, for example. It is preferable.
  • (L 2 ) for example, the distance between the first region (D1) and the second region (D2) can be sufficiently maintained. For this reason, for example, the formation of a G-quartet structure by the first region (D1) and the second region (D2) in the absence of the target is sufficiently suppressed, and the catalytic function in the absence of the target is achieved. And the background based on fluorescence can be sufficiently reduced.
  • the nucleic acid molecule (II) can be represented by, for example, D1-W-D2, and specifically can be represented by the following formula (I).
  • 5 'side sequence (N) n1 -GGG- (N) n2 - (N) n3 - is the sequence of the first region (D1) (d1)
  • 3 ′ sequence-(N) m3- (N) m2 -GGG- (N) m1 is the sequence (d2) of the second region (D2)
  • W is a region between the first region (D1) and the second region (D2), including the coupling region (A)
  • N represents a base
  • n1, n2, and n3, and m1, m2, and m3 represent the number of repetitions of the base N, respectively.
  • Formula (I) shows a state in which the first region (D1) and the second region (D2) are aligned in the nucleic acid molecule (II), which is the first region (D1).
  • the second region (D2) are schematic views showing the arrangement relationship between the first region (D1) and the second region (D2) in the present invention. This is not a limitation.
  • (N) n1 and (N) m1 satisfy the following condition (1): N) n2 and (N) m2 preferably satisfy the following condition (2), and (N) n3 and (N) m3 preferably satisfy the following condition (3).
  • Condition (1) In (N) n1 and (N) m1 , the base sequence from the 5 ′ end of (N) n1 and the base sequence from the 3 ′ end of (N) m1 are complementary to each other, and n1 and m1 Are the same 0 or a positive integer.
  • Condition (2) (N) n2 and (N) m2 are such that the base sequence from the 5 ′ end of (N) n2 and the base sequence from the 3 ′ end of (N) m2 are non-complementary to each other, m2 is a positive integer, and may be the same or different.
  • (N) n3 and (N) m3 are those in which n3 and m3 are 3 or 4, respectively, and may be the same or different, have three bases G, and when n3 or m3 is 4, (N) n3 and (N) m3, the second or third base is a base H except G.
  • the condition (1) is a condition of (N) n1 at the 5 ′ end and (N) m1 at the 3 ′ end when the first region (D1) and the second region (D2) are aligned. .
  • the base sequence from the 5 ′ end of (N) n1 and the base sequence from the 3 ′ end of (N) m1 are complementary to each other and have the same length.
  • (N) n1 and (N) m1 are complementary sequences of the same length, they can be said to be stem regions that form stems in an aligned state.
  • N1 and m1 may be the same 0 or a positive integer, and are, for example, 0, 1 to 10, and preferably 1, 2, or 3, respectively.
  • the condition (2) is a condition of (N) n2 and (N) m2 when the first region (D1) and the second region (D2) are aligned.
  • the base sequence of (N) n2 and the base sequence of (N) m2 are non-complementary to each other, and n2 and m2 may have the same length or different lengths. Since (N) n2 and (N) m2 are non-complementary sequences, they can be said to be regions that form an inner loop in an aligned state.
  • N2 and m2 are positive integers, for example, 1 to 10 respectively, preferably 1 or 2.
  • n2 and m2 may be the same or different.
  • n2 m2, n2> m2, and n2 ⁇ m2, and preferably n2> m2 and n2 ⁇ m2.
  • the condition (3) is a condition of (N) n3 and (N) m3 when the first region (D1) and the second region (D2) are aligned.
  • the base sequence of (N) n3 and the base sequence of (N) m3 are 3 or 4 base length sequences having 3 bases G, and the same or different May be.
  • n3 or m3 is 4,
  • (N) n3 and (N) m3 are bases H other than G in the second or third base.
  • Examples of the base H that is a base other than G include A, C, T, and U, and preferably A, C, or T.
  • condition (3) include the following conditions (3-1), (3-2), and (3-3).
  • Condition (3-1) Among (N) n3 and (N) m3 , the sequence from one 5 ′ side is GHGG, and the sequence from the other 5 ′ side is GGG.
  • Condition (3-2) Among (N) n3 and (N) m3 , the sequence from one 5 ′ side is GGHG, and the sequence from the other 5 ′ side is GGG.
  • Condition (3-3) Both (N) n3 and (N) m3 sequences are GGG.
  • the binding region (A) is, for example, at least one polynucleotide selected from the group consisting of (a1) to (a4), wherein the polynucleotide of (a1) is This is the case of a polynucleotide comprising a base sequence surrounded by a square in any of the base sequences of SEQ ID NOS: 280 to 299 in Table 2, and the description thereof can be incorporated.
  • one of the first region (D1) and the second region (D2) is, for example, in at least one polynucleotide selected from the group consisting of (e1) to (e4)
  • the polynucleotide of (e1) is represented by SEQ ID NOs: 280 to 299 in Table 2 above. It is a polynucleotide that has been replaced with a polynucleotide comprising an underlined base sequence in any base sequence, and the description can be used after the above replacement.
  • the length of the first region (D1) is not particularly limited, and the lower limit is, for example, 7 base length, 8 base length, 10 base length, and the upper limit is, for example, 30 base length, 20 base length, 10
  • the base length and the range thereof are, for example, 7 to 30 base length, 7 to 20 base length, and 7 to 10 base length.
  • the length of the second region (D2) is not particularly limited, and the lower limit is, for example, 7 base length, 8 base length, 10 base length, and the upper limit is, for example, 30 base length, 20 base length, 10
  • the base length and the range thereof are, for example, 7 to 30 base length, 7 to 20 base length, and 7 to 10 base length.
  • the lengths of the first region (D1) and the second region (D2) may be the same or different.
  • the nucleic acid molecule (II) is, for example, at least one polynucleotide selected from the group consisting of the following (t1) to (t3) and (t4).
  • T1 A polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 280 to 299 (t2) In any one of the nucleotide sequences of (t1), one or several bases are deleted, substituted, inserted and / or added.
  • “having the same function as (t1)” means that in the absence of the target, the G formation region (D) does not form a G-quartet structure, and in the presence of the target. This means that the target is bonded to the bonding region (A) and the G-forming region (D) forms a G-quartet structure.
  • the polynucleotide (t1) is a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 280 to 299.
  • “1 or several” means, for example, 1 to 10, 1 to 7, 1 to 5, 1 to 5 in any base sequence of (t1). Three, one or two.
  • identity is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide (t1).
  • “hybridization” and “stringent conditions” the above description can be used.
  • the polynucleotides (t2) to (t4) preferably have at least one base sequence of the binding region (A) and the G-forming region (D) conserved in the base sequence of each SEQ ID NO. .
  • the conserved base sequence is, for example, aligned based on the base sequence of each SEQ ID NO of the polynucleotide of (t1), and the corresponding base sequence is the conserved base It can be judged as an array.
  • the identity is, for example, 50% or more, 60% or more, 70% or more, 80% or more 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the length of the nucleic acid molecule (II) is not particularly limited.
  • the lower limit of the length of the nucleic acid molecule (II) is, for example, 25 base length, 30 base length, 35 base length, and the upper limit is, for example, 200 base length, 100 base length, 80 base length, the range is For example, it is 25 to 200 bases long, 30 to 100 bases long, 35 to 80 bases long.
  • the nucleic acid molecule (III) is a double-stranded nucleic acid molecule composed of a first strand (ss1) and a second strand (ss2),
  • the first strand (ss1) has the G-forming region (D) and the binding region (A) in this order
  • the second chain (ss2) has a stem formation region (S D ) and a stem formation region (S A ) in this order, and the stem formation region (S D ) is in the G formation region (D).
  • the stem-forming region (S A ) is a double-stranded nucleic acid molecule having a sequence complementary to the binding region (A).
  • the G-forming region (D) is, for example, the single-stranded type.
  • the G-quartet formation of the G-forming region (D) is controlled to be ON-OFF depending on the presence or absence of a target based on the following mechanism. Note that the present invention is not limited to this mechanism.
  • the nucleic acid molecule (III) anneals the G-forming region (D) of the first strand (ss1) and the stem-forming region (S D ) of the second strand (ss2).
  • formation of the G-quartet structure in the G-forming region (D) is inhibited (switch-OFF), and as a result, for example, formation of a complex between the G-forming region (D) and porphyrin is inhibited.
  • the binding region (A) of the first strand (ss1) and the stem formation region (S A ) of the second strand (ss2) are annealed, whereby the binding region (A ) Structure is also fixed.
  • the structure of the molecule in this state is also called an inactive type.
  • the nucleic acid molecule (III) is, under the target presence, by contact of the target to the binding region (A), annealing of the coupling region (A) and the stem forming regions (S A) is released The three-dimensional structure of the binding region (A) changes to a more stable structure.
  • the annealing of the G formation region (D) and the stem formation region (S D ) is canceled, and a G-quartet structure is formed in the region of the G formation region (D) (switch-ON).
  • a complex of the G-forming region (D) and porphyrin is formed, causing a catalytic function and fluorescence.
  • the structure of the molecule in this state is also called an active form.
  • the nucleic acid molecule (III) does not cause the catalytic function and fluorescence due to the complex formation in the absence of the target, and does not cause the catalytic function and fluorescence due to the complex formation only in the presence of the target. Therefore, target analysis such as qualitative or quantitative is possible.
  • the stem formation region (S D ) preferably has a sequence that is entirely or partially complementary to a part of the G formation region (D).
  • the stem forming region (S A ) is, for example, a sequence that is entirely or partially complementary to a part of the binding region (A).
  • the sequence of each region, the G forming region (D) and the stem forming region and the (S D) is annealed, the binding region (A) and the stem forming regions (S A ) And the annealing order.
  • the following order can be illustrated as a specific example. (1) ss1 5'- AD-3 ' ss2 3'- S A -S D -5 ' (2) ss1 5'- DA-3 ' ss2 3'- S D -S A -5 '
  • the stem formation region (S A ) is complementary to the 3′-side region of the binding region (A), and the stem formation region (S D ) is the G formation region (D). It is preferable to be complementary to the 5 ′ side region.
  • the stem formation region (S D ) is complementary to the 3′-side region of the G formation region (D), and the stem formation region (S A ) is the binding region (A). It is preferable to be complementary to the 5 ′ side region.
  • the regions may be connected directly or indirectly.
  • the direct connection means that, for example, the 3 ′ end of one region and the 5 ′ end of the other region are directly bonded, and the indirect connection is, for example, 3 of one region. It means that the “end” and the 5 ′ end of the other region are bound via the intervening linker region.
  • the intervening linker region may be, for example, a nucleic acid sequence or a non-nucleic acid sequence, preferably the former.
  • the nucleic acid molecule (III) is, for example, between the binding region (A) and the G-forming region (D) in the first strand (ss1) and the stem-forming region in the second strand (ss2). It is preferable to have the intervening linker region between (S D ) and the stem forming region (S A ).
  • the intervening linker region (L 1 ) in the first strand (ss1) and the intervening linker region (L 2 ) in the second strand (ss2) are preferably non-complementary sequences.
  • an intervening linker region that connects the binding region (A) and the G-forming region (D) is (L 1 ), the stem-forming region (S D ), and the stem-forming region (S A )
  • the intervening linker region linking is represented by (L 2 ).
  • the nucleic acid molecule (III) may have both (L 1 ) and (L 2 ) as an intervening linker region, or may have only one of them.
  • the formation of the G-quartet structure is turned on and off as follows.
  • the binding region (A), the stem formation region (S A ), the G formation region (D), and the stem formation region (S D ) form stems, respectively.
  • the intervening linker region (L 1 ) and the intervening linker region (L 2 ) form an internal loop that inhibits the formation of the G-quartet structure of the G-forming region (D).
  • the respective stem formation is released by the contact of the target with the binding region (A), and a G-quartet structure is formed in the G formation region (D).
  • the lengths of the stem-forming sequence (S A ) and the stem-forming sequence (S D ) are not particularly limited.
  • the length of the stem forming sequence (S A ) is, for example, 1 to 60 bases long, 1 to 10 bases long, or 1 to 7 bases long.
  • the stem-forming sequence (S D ) has a length of, for example, 1 to 30 bases, 0 to 10 bases, 1 to 10 bases, 0 to 7 bases, or 1 to 7 bases.
  • the stem forming sequence (S A ) and the stem forming sequence (S D ) may have the same length, the former may be long, or the latter may be long.
  • the lengths of the intervening linker regions (L 1 ) and (L 2 ) are not particularly limited.
  • the lengths of the intervening linker regions (L 1 ) and (L 2 ) are, for example, 0 to 30 bases, 1 to 30 bases, 1 to 15 bases, and 1 to 6 bases, respectively.
  • the lengths of the intervening linker regions (L 1 ) and (L 2 ) may be the same or different, for example. In the latter case, the difference in length of the intervening linker region (L 1) and (L 2) is not particularly limited, for example, 1 to 10 bases in length, 1 or 2 bases in length, one base in length.
  • the binding region (A) is, for example, at least one polynucleotide selected from the group consisting of (a1) to (a4), wherein the polynucleotide of (a1) is This is the case of a polynucleotide comprising a base sequence surrounded by a square in any one of SEQ ID NOS: 300 to 427 in Tables 3A to D, and the description thereof can be used.
  • the G-forming region (D) is, for example, at least one polynucleotide selected from the group consisting of (d1) to (d4), wherein the polynucleotide of (d1) is This is a case of a polynucleotide comprising the underlined base sequence in any of the base sequences of SEQ ID NOS: 300 to 427 in Tables 3A to D, and the description thereof can be used.
  • the first strand (ss1) is, for example, at least one polynucleotide selected from the group consisting of (u1) to (u3) and (u4) below.
  • (U1) A polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 300 to 427 (u2) In any one of the nucleotide sequences of (u1), one or several bases are deleted, substituted, inserted and / or added.
  • “having the same function as (u1)” means that in the absence of the target, the G formation region (D) does not form a G-quartet structure, and in the presence of the target. This means that the target is bonded to the bonding region (A) and the G-forming region (D) forms a G-quartet structure.
  • the polynucleotide (u1) is a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 300 to 427.
  • “1 or several” means, for example, 1 to 10, 1 to 7, 1 to 5, 1 to 5 in any one of the nucleotide sequences of (u1). Three, one or two.
  • identity is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide (u1).
  • “hybridization” and “stringent conditions” the above description can be used.
  • the conserved base sequence is, for example, aligned based on the base sequence of each SEQ ID NO of the polynucleotide (u1), and the corresponding base sequence is the conserved base. It can be judged as an array.
  • the identity is, for example, 50% or more, 60% or more, 70% or more, 80% or more 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the second strand (ss2) is, for example, at least one polynucleotide selected from the group consisting of the following (v1) to (v3) and (v4).
  • V1 A polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 428 to 555 (v2) In the nucleotide sequence of any one of (v1), one or several bases are deleted, substituted, inserted and / or added.
  • a polynucleotide having a function equivalent to that of (v4) From a nucleotide sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences of (v1) above A polynucleotide having the same function as (v1) above
  • “having the same function as (v1)” means that the G-forming region (D) has a G-quartet structure by annealing with the first strand (ss1) in the absence of the target. In the presence of the target, the target binds to the binding region (A), annealing with the first strand (ss1) is released, and the G-forming region (D) becomes G- It means forming a quartet structure.
  • the polynucleotide (v1) is a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 428 to 555 in Tables 4A and B below.
  • “1 or several” means, for example, any one of the nucleotide sequences of (v1), for example, 1 to 10, 1 to 7, 1 to 5, 1 to Three, one or two.
  • identity is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide (v1).
  • “hybridization” and “stringent conditions” the above description can be used.
  • the combination of the first strand (ss1) and the second strand (ss2) of the nucleic acid molecule (III) is not particularly limited, and includes, for example, (1) to (127) and (128) in Table 5 below. It is at least one combination selected from the group, and each of the first strand (ss1) and the second strand (ss2) includes a polynucleotide having a base sequence having a sequence number corresponding to the combination.
  • the polynucleotide of (u1) may have the sequence number corresponding to the combination.
  • the polynucleotide may be replaced with a polynucleotide having a base sequence, and the description can be used after the above replacement.
  • the polynucleotide of (v1) has the sequence number corresponding to the combination.
  • the polynucleotide may be replaced with a polynucleotide having a base sequence, and the description can be used after the above replacement.
  • the lengths of the first strand (ss1) and the second strand (ss2) are not particularly limited.
  • the length of the first strand (ss1) is, for example, 40 to 200 bases long, 42 to 100 bases long, 45 to 60 bases long.
  • the length of the second strand (ss2) is, for example, 4 to 120 bases long, 5 to 25 bases long, or 10 to 15 bases long.
  • the nucleic acid molecule (III) includes, for example, a third strand (ss3), and the third strand has a stem forming region (S ′ D ) and a stem forming region (S ′ A ) in this order,
  • the stem formation region (S ′ D ) has a sequence complementary to the G formation region (D), and the stem formation region (S ′ A ) is complementary to the binding region (A). May have a different arrangement.
  • the third strand (ss3) can bind to, for example, the binding region (A) and the G-forming region (D) of the two first strands (ss1). That is, the third chain (ss3) can cross-link two first chains (ss1), for example.
  • the third strand (ss3) detects a target by, for example, crosslinking a plurality of first strands (ss1).
  • the first strand (ss1) can be integrated, and the sensitivity of the sensor can be improved.
  • a molecule in which a plurality of nucleic acid molecules (III) (first strand (ss1)) are cross-linked by the third strand (ss3) is also referred to as a nucleic acid complex (III).
  • the order of the regions is such that the G-forming region (D) and the stem-forming region (S ′ D ) are annealed, and the binding region (A ) And the stem forming region (S ′ A ) are annealed, and the first strand (ss1) in which the stem forming region (S ′ D ) is annealed and the stem forming region (S ′ A ) are annealed.
  • What is necessary is just the order from which the 1st chain
  • the stem formation region (S ′ A ) is complementary to the 5 ′ side region of the binding region (A), and the stem formation region (S ′ D ) is the G formation region ( It is preferably complementary to the 3 ′ region of D).
  • the stem formation region (S ′ D ) is complementary to the 5 ′ side region of the G formation region (D), and the stem formation region (S ′ A ) It is preferably complementary to the 3 ′ region of A).
  • the third chain (ss3) may be connected directly or indirectly between the regions, for example.
  • the above description can be used, for example.
  • the third strand (ss3) preferably has, for example, the intervening linker region (L 3 ) between the stem forming region (S ′ A ) and the stem forming region (S ′ D ).
  • the intervening linker region (L 3 ) and, for example, the intervening linker regions (L 1 ) and (L 2 ) are preferably non-complementary sequences.
  • an intervening linker region that connects the binding region (A) and the G-forming region (D) is (L 1 ), the stem-forming region (S ′ D ), and the stem-forming region (S ′ A And an intervening linker region linking with (L 3 ).
  • the nucleic acid molecule (III) may have, for example, any one of (L 1 ), (L 2 ) and (L 3 ) as an intervening linker region, or two or more. Or you may have everything.
  • the lengths of the stem-forming sequence (S ′ A ) and the stem-forming sequence (S ′ D ) are not particularly limited.
  • the length of the stem forming sequence (S ′ A ) is, for example, 1 to 60 bases long, 1 to 10 bases long, or 1 to 7 bases long.
  • the length of the stem forming sequence (S ′ D ) is, for example, 1 to 30 bases long, 1 to 10 bases long, or 1 to 7 bases long.
  • the stem forming sequence (S ′ A ) and the stem forming sequence (S ′ D ) may have the same length, the former may be long, or the latter may be long.
  • the length of the intervening linker region (L 3 ) is not particularly limited.
  • the length of the intervening linker region (L 3 ) is, for example, 1 to 30 bases long, 1 to 15 bases long, or 1 to 6 bases long.
  • the length of the intervening linker region (L 3 ) may be the same as or different from the length of the intervening linker regions (L 1 ) and (L 2 ), for example.
  • the number of the first strand (ss1) and the second strand (ss2) included in the nucleic acid complex (III) is not particularly limited.
  • the first strand (ss1) and the second strand (ss2) may be linked directly or indirectly.
  • the nucleic acid molecule (III) can be referred to as a single-stranded nucleic acid sensor, for example, and the first strand (ss1) ) And the second strand (ss2) can be referred to as a first region and a second region, respectively.
  • the direct connection means that, for example, the 3 ′ end of one region and the 5 ′ end of the other region are directly bonded, and the indirect connection is, for example, 3 of one region.
  • the intervening linker region may be, for example, a nucleic acid sequence or a non-nucleic acid sequence, preferably the former.
  • the length of the intervening linker region is not particularly limited and is, for example, 1 to 60 bases long.
  • the order of the regions in the nucleic acid molecule (III) is the G-forming region (D) and the stem-forming region.
  • S D may be annealed and the bonding region (A) and the stem formation region (S A ) may be annealed.
  • the following order can be illustrated as a specific example. (5) 5'- AD S D -S A -3 ' (6) 5'-S D -S A -AD -3 ' (7) 5'- A-D- L 1 -S D -S A -3 ' (8) 5'-S D -S A -L 1 -AD -3 '
  • the stem formation region (S A ) is complementary to the 3′-side region of the binding region (A), and the stem formation region (S D ) It is preferably complementary to the 5 ′ region of the region (D).
  • the stem formation region (S D ) is complementary to the 3′-side region of the G formation region (D), and the stem formation region (S A ) It is preferably complementary to the 5 ′ region of the region (A).
  • the regions may be connected directly or indirectly.
  • the direct connection means that, for example, the 3 ′ end of one region and the 5 ′ end of the other region are directly bonded, and the indirect connection is, for example, 3 of one region. It means that the “end” and the 5 ′ end of the other region are bound via the intervening linker region.
  • the intervening linker region may be, for example, a nucleic acid sequence or a non-nucleic acid sequence, preferably the former.
  • the nucleic acid molecule (III) is, for example, the binding region (A) in the first region and the G-forming region ( It is preferable that the intervening linker region is provided between the intermediate region D) and between the stem formation region (S D ) and the stem formation region (S A ) in the second region.
  • the intervening linker region (L 2 ) in the first region and the intervening linker region (L 3 ) in the second region are preferably non-complementary sequences.
  • an intervening linker region that connects the binding region (A) and the G-forming region (D) is (L 2 ), the stem-forming region (S D ), and the stem-forming region (S A )
  • the intervening linker region linking is represented by (L 3 ).
  • the nucleic acid molecule (III) can be used as, for example, an intervening linker region of (L 2 ) and (L 3 ). You may have both, and you may have only any one.
  • the formation of the G-quartet structure is turned on and off as follows.
  • the binding region (A), the stem formation region (S A ), the G formation region (D), and the stem formation region (S D ) form stems, respectively.
  • the intervening linker region (L 2 ) and the intervening linker region (L 3 ) form an internal loop that inhibits the formation of the G-quartet structure of the G-forming region (D).
  • the respective stem formation is released by the contact of the target with the binding region (A), and a G-quartet structure is formed in the G formation region (D).
  • the length of the stem-forming sequence (S A ) and the stem-forming sequence (S D ) in the nucleic acid molecule (III) is not particularly limited, and for example, the above description can be used.
  • the lengths of the intervening linker regions (L 2 ) and (L 3 ) are not particularly limited.
  • the lengths of the intervening linker regions (L 2 ) and (L 3 ) are, for example, 0 to 30 bases, 1 to 30 bases, 1 to 15 bases, and 1 to 6 bases, respectively.
  • the lengths of the intervening linker regions (L 2 ) and (L 3 ) may be the same or different, for example. In the latter case, the difference in length between the intervening linker regions (L 2 ) and (L 3 ) is not particularly limited, and is, for example, 1 to 10 bases long, 1 or 2 bases long, and 1 base long.
  • the sensor of the present invention may be, for example, a sensor including the nucleic acid molecule or a sensor composed of the nucleic acid molecule.
  • the sensor of the present invention is a molecule containing a nucleotide residue, and may be, for example, a molecule consisting only of a nucleotide residue or a molecule containing a nucleotide residue.
  • the nucleotide is, for example, ribonucleotide, deoxyribonucleotide and derivatives thereof.
  • the sensor may be, for example, DNA containing deoxyribonucleotide and / or a derivative thereof, RNA containing ribonucleotide and / or a derivative thereof, or a chimera (DNA / RNA) containing the former and the latter But you can.
  • the sensor is preferably DNA.
  • the nucleotide may contain, for example, either a natural base (non-artificial base) or a non-natural base (artificial base) as a base.
  • a natural base include A, C, G, T, U, and modified bases thereof.
  • the modification include methylation, fluorination, amination, and thiolation.
  • the unnatural base include 2′-fluoropyrimidine, 2′-O-methylpyrimidine and the like. Specific examples include 2′-fluorouracil, 2′-aminouracil, 2′-O-methyluracil, And 2'-thiouracil.
  • the nucleotide may be, for example, a modified nucleotide, and the modified nucleotide is, for example, a 2′-methylated-uracil nucleotide residue, 2′-methylated-cytosine nucleotide residue, 2′-fluorinated-uracil nucleotide. Residue, 2′-fluorinated-cytosine nucleotide residue, 2′-aminated-uracil nucleotide residue, 2′-aminated-cytosine nucleotide residue, 2′-thiolated-uracil nucleotide residue, 2′- Thio-cytosine nucleotide residues and the like.
  • the nucleic acid molecule may include non-nucleotides such as PNA (peptide nucleic acid) and LNA (Locked Nucleic Acid), for example.
  • the sensor of the present invention may further include, for example, a linker region (L), and the linker region (L) is preferably linked to the end of the nucleic acid molecule.
  • the linker region (L) may be linked to, for example, at least one or both of the 3 'end and the 5' end of the nucleic acid molecule, and is preferably linked to the 3 'end of the nucleic acid molecule.
  • the sensor of the present invention When the sensor of the present invention has the linker region (L), it may be immobilized on a carrier or the like by the linker region (L).
  • the analytical sensor of the present invention may be immobilized, for example, at the 3 ′ end or 5 ′ end of the linker region (L).
  • the linker region (L ) Are preferably immobilized at the 3 ′ end of the linker region (L).
  • the length of the linker region (L) is not particularly limited, and the lower limit is, for example, 1 base length, 3 base lengths, 5 base lengths, and the upper limit is, for example, 200 base length, 50 base length, 20 base length, 12 base length, 9 base length, the range is, for example, 1-200 base length, 1-50 base length, 1-20 base length, 3-12 base It is 5-9 bases long.
  • the linker region (L) is, for example, a nucleotide or a polynucleotide, and the structural unit is, for example, a nucleotide residue.
  • the above-mentioned illustration can be used for the said nucleotide residue, for example.
  • the linker region (L) is not particularly limited, and examples thereof include polynucleotides such as DNA consisting of deoxyribonucleotide residues and DNA containing ribonucleotide residues.
  • linker examples include polydeoxythymine (poly dT), polydeoxyadenine (poly dA), poly dAdT which is a repeating sequence of A and T, and preferably poly dT and poly dAdT.
  • the nucleic acid molecule may be, for example, a single-stranded polynucleotide.
  • the single-stranded polynucleotide is preferably capable of forming a stem structure and a loop structure by, for example, self-annealing. Specifically, for example, a stem loop structure, an internal loop structure and / or a bulge structure can be formed. It is preferable that
  • the stem structure and the loop structure can be formed means, for example, that the stem structure and the loop structure are actually formed, and even if the stem structure and the loop structure are not formed, the stem structure depending on the conditions. And the ability to form a loop structure.
  • a stem structure and a loop structure can be formed includes, for example, both experimental confirmation and prediction by a computer simulation.
  • the nucleic acid molecule is preferably nuclease resistant, for example.
  • the nucleic acid molecule preferably has, for example, the modified nucleotide residue and / or the artificial nucleic acid monomer residue for nuclease resistance.
  • the nucleic acid molecule is resistant to nuclease, for example, tens of kDa PEG (polyethylene glycol) or deoxythymidine may be bound to the 5 'end or 3' end.
  • the sensor of the present invention preferably further includes, for example, a carrier, and the nucleic acid molecule is immobilized on the carrier.
  • the nucleic acid molecule can be directly or indirectly immobilized at, for example, either the 3 ′ end or the 5 ′ end, and the indirect In the case of immobilization, the nucleic acid molecule can be immobilized with, for example, the linker region.
  • the nucleic acid molecule immobilization method is not particularly limited, and can be performed by, for example, a known nucleic acid immobilization method.
  • the sensor of the present invention may further include a reagent that reacts with the G-forming region (D), for example.
  • the reagent preferably contains, for example, porphyrin that forms a complex with the G-forming region (D) having a G-quartet structure.
  • porphyrin for example, the above description can be used.
  • the senor of the present invention can set the reagent depending on, for example, which function of the G formation region (D) is used in target analysis.
  • the analytical sensor of the present invention preferably contains, for example, a substrate for the oxidation-reduction reaction as the reagent, and includes both the porphyrin and the substrate. May be included.
  • the substrate is not particularly limited, and for example, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), 1,2-phenylenediamine (OPD), 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfoniconAcid ) MmAmmonium Salt (ABTS), 3,3′-Diaminobenzodinine (DAB), 3,3′-Diaminobenzodinine Tetrahydrochloride Hydrate (DAB4HCl), 3-Amino-9-ethylCarbolC1) 2,4,6-Tribromo-3-hydroxybenzoic acid, 2, -Dichlorophenol, 4-Aminoantipyrine, 4-Aminoantipyrine Hydrochloride, luminol and the like.
  • TMB 5,5′-tetramethylbenzidine
  • OPD 1,2-phenylenediamine
  • 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfoniconAcid ) MmAmmonium Salt
  • the nucleic acid molecule may further have a labeling substance and may be labeled with the labeling substance.
  • the labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, isotopes and enzymes.
  • the fluorescent substance include fluorophores such as pyrene, TAMRA, fluorescein, Cy (registered trademark) 3 dye, Cy (registered trademark) 5 dye, FAM dye, rhodamine dye, Texas red dye, JOE, MAX, HEX, and TYE.
  • the dye include Alexa dyes such as Alexa (registered trademark) 488 and Alexa (registered trademark) 647.
  • the enzyme include luciferase.
  • the labeling substance may be linked directly to the nucleic acid molecule or indirectly via the linker region (L), for example.
  • cortisol in a sample can be detected.
  • the analysis method of the present invention combines the contact step of bringing a sample into contact with the cortisol analysis sensor of the present invention, and combining the cortisol in the sample and the binding region (A) in the cortisol analysis sensor. And a detection step of detecting cortisol in the sample.
  • the analysis method of the present invention is characterized by using the sensor of the present invention, and other processes and conditions are not particularly limited.
  • cortisol specifically binds to the binding region (A) of the sensor of the present invention, and the G-forming region of the sensor of the present invention when cortisol is bound.
  • (D) forms a G-quartet structure and becomes active, for example, by detecting the binding between cortisol and the sensor using the active nature of the G-forming region (D), Cortisol in the sample can be specifically analyzed. Specifically, for example, since the presence or absence of cortisol or the amount of cortisol in a sample can be analyzed, it can be said that qualitative or quantitative determination is also possible.
  • the sample is not particularly limited.
  • the sample include a biological sample.
  • the biological sample include blood, serum, plasma, interstitial fluid, urine, saliva, sweat, tears, and runny nose.
  • the sample may be, for example, a liquid sample or a solid sample.
  • the sample is preferably a liquid sample because it is easy to contact with the nucleic acid molecule and is easy to handle.
  • a mixed solution, an extract, a dissolved solution, and the like may be prepared using a solvent and used.
  • the solvent is not particularly limited, and examples thereof include water, physiological saline, and buffer solution.
  • the method for contacting the sample with the analytical sensor is not particularly limited.
  • the contact between the sample and the analysis sensor is preferably performed in a liquid, for example.
  • the liquid is not particularly limited, and examples thereof include water, physiological saline, and buffer solution.
  • the contact condition between the sample and the sensor is not particularly limited.
  • the contact temperature is, for example, 4 to 37 ° C. or 18 to 25 ° C.
  • the contact time is, for example, 10 to 120 minutes or 30 to 60 minutes.
  • the senor may be, for example, an immobilized sensor in which the nucleic acid molecule is immobilized on a carrier, or an unfixed sensor in which the nucleic acid molecule is not immobilized and released. In the latter case, for example, the sample and the non-fixed sensor are brought into contact in a container.
  • the sensor is preferably, for example, the immobilized sensor because of excellent handling properties.
  • the carrier is not particularly limited, and examples thereof include a substrate, a bead, and a container. Examples of the container include a microplate and a tube.
  • the immobilization of the nucleic acid molecule in the sensor is, for example, as described above.
  • the detection step is a step of detecting the binding between cortisol in the sample and the binding region (A) in the sensor.
  • the detection step may further include, for example, a step of analyzing the presence or amount of cortisol in the sample based on the result of the detection step.
  • a step of analyzing the presence or amount of cortisol in the sample based on the result of the detection step.
  • the binding between the cortisol and the binding region (A) in the sensor cannot be detected, it can be determined that cortisol is not present in the sample, and if the binding is detected, It can be judged that cortisol is present. Further, based on the binding amount obtained in the detection step, the amount of the cortisol, and the correlation between the binding amounts of the two, for example, the amount of cortisol in the sample can be calculated.
  • the method for detecting the binding between the cortisol and the binding region (A) in the sensor is not particularly limited.
  • the active function of the G-forming region (D) linked to the binding region (A) Can be given.
  • the active function is not particularly limited, and examples thereof include the catalytic function of the G-forming region (D) and the fluorescence of the G-forming region (D) as described above.
  • the analysis reagent of the present invention includes the sensor for cortisol analysis of the present invention.
  • the analysis kit of the present invention includes the sensor for analyzing cortisol of the present invention.
  • the analysis reagent and analysis kit of the present invention only need to include the sensor for cortisol analysis of the present invention, and other configurations and conditions are not particularly limited. If the analysis reagent and analysis kit of the present invention are used, for example, cortisol can be detected as described above.
  • the analysis reagent and analysis kit of the present invention can be used, for example, in the stress evaluation method of the present invention described later and the method for testing the possibility of cortisol-related diseases of the present invention. Therefore, the analysis reagent can also be referred to as, for example, a stress evaluation reagent or a test reagent (diagnostic reagent) for cortisol-related diseases.
  • the analysis kit can also be referred to as, for example, a stress evaluation kit or a cortisol-related disease test kit (diagnostic kit).
  • the analysis reagent and analysis kit of the present invention may further contain a reagent that reacts with the G-forming region (D) of the sensor for cortisol analysis, for example.
  • a reagent that reacts with the G-forming region (D) of the sensor for cortisol analysis
  • the reagent include a porphyrin forming a complex with the G-forming region (D) having a G-quartet structure, a substrate for the catalytic function of the G-forming region (D) having a G-quartet structure, and the like. It is done.
  • the porphyrin for example, the above description can be used.
  • the analysis kit of the present invention may include other components in addition to the sensor of the present invention, for example.
  • the component include the carrier, the reagent, a buffer solution, and instructions for use.
  • the description of the sensor of the present invention can be used for the analysis reagent and analysis kit of the present invention, and the description of the sensor of the present invention and the analysis method of the present invention can also be used for the method of use thereof.
  • the stress evaluation reagent of the present invention includes the sensor for cortisol analysis of the present invention.
  • the stress evaluation kit of the present invention comprises the sensor for cortisol analysis of the present invention.
  • the evaluation reagent and the evaluation kit of the present invention only need to include the sensor for cortisol analysis of the present invention, and other configurations and conditions are not particularly limited. If the evaluation reagent and evaluation kit of the present invention are used, for example, stress evaluation can be performed as described later.
  • the description of the sensor, analysis reagent, and analysis kit of the present invention can be used, and the sensor, analysis reagent, analysis kit of the present invention, The description of the evaluation method of the present invention to be described later can be cited.
  • the stress evaluation method of the present invention includes a contact step of bringing a sample of a subject into contact with the sensor for cortisol analysis of the present invention, and the binding region (cortisol in the sample and the sensor for cortisol analysis) A detection step of detecting cortisol in the sample by combining with A), and an acquisition step of acquiring information on stress by comparing the cortisol amount in the detection step with a reference value Features.
  • the evaluation method of the present invention is characterized by using the sensor of the present invention, and other processes and conditions are not particularly limited.
  • the description of the sensor of the present invention and the analysis method of the present invention can be cited.
  • the description of the analysis method of the present invention can be used for the contact step and the detection step.
  • Examples of the subject include humans, non-human animals other than humans, and the non-human animals include, for example, mammals such as mice, rats, dogs, monkeys, rabbits, sheep, and horses, as described above. Can be given.
  • the reference value can be obtained using, for example, a sample isolated from a healthy person who is not stressed (unloaded state) and / or is stressed (loaded state).
  • the reference value may be measured simultaneously with the sample of the subject or may be measured in advance.
  • the unloaded state and the loaded state can be implemented by, for example, a known stress test.
  • a method for acquiring information related to the stress of the subject is not particularly limited, and can be appropriately determined according to the type of the reference value.
  • the amount of cortisol in the sample of the subject is higher than the amount of cortisol in the sample of the unloaded healthy subject, the case is the same as the amount of cortisol in the sample of the healthy healthy subject (significant difference) And / or if it is significantly higher than the amount of cortisol in the sample of the healthy subject under load, the subject can obtain information that it is in a stress state.
  • the subject when the amount of cortisol in the subject's sample is lower than the amount of cortisol in the unloaded healthy sample, the same amount as the cortisol in the unloaded healthy sample (significant difference And / or if the amount is significantly lower than the amount of cortisol in a sample of a healthy subject under load, the subject can obtain information that the subject is unstressed.
  • the test reagent (diagnostic reagent) for cortisol-related disease of the present invention includes the sensor for cortisol analysis of the present invention.
  • the test kit (diagnostic kit) for cortisol-related diseases of the present invention comprises the sensor for cortisol analysis of the present invention.
  • the test reagent and test kit of the present invention only need to include the sensor for cortisol analysis of the present invention, and other configurations and conditions are not particularly limited. If the test reagent and test kit of the present invention are used, for example, the possibility of morbidity of cortisol-related diseases can be tested as described later.
  • test reagent and test kit of the present invention for example, the description of the sensor, analysis reagent, and analysis kit of the present invention can be used, and the sensor, analysis reagent, analysis kit of the present invention, The description of the test method of the present invention described later can be cited.
  • the method for testing the morbidity of a cortisol-related disease includes a contact step of bringing a sample of a subject into contact with the sensor for cortisol analysis according to the present invention, and cortisol in the sample and the cortisol.
  • a contact step of bringing a sample of a subject into contact with the sensor for cortisol analysis according to the present invention, and cortisol in the sample and the cortisol.
  • test method of the present invention for example, the description of the sensor of the present invention and the analysis method and evaluation method of the present invention can be used.
  • the description of the analysis method of the present invention can be used for the contact step and the detection step.
  • cortisol-related disease for example, the possibility of the onset of cortisol-related disease (hereinafter also referred to as “related disease”), the presence or absence of the onset of the related disease, the degree of progression of the related disease and the prognostic state can be evaluated.
  • the related disease is, for example, a disease caused by an increase or decrease in the cortisol.
  • the related diseases caused by the decrease in cortisol include, for example, Addison's disease, congenital adrenal hypoplasia, congenital adrenal hyperplasia, pituitary tumor, pituitary hypoadrenocorticism, hypothalamus sexual adrenocortical hypofunction and the like.
  • the related diseases caused by the increase in cortisol include Cushing disease, Cushing syndrome, glucocorticoid refractory disease and the like.
  • the reference value is not particularly limited, and examples thereof include the amount of cortisol in healthy subjects, related disease patients, and related disease patients for each stage of related diseases.
  • the reference value may be, for example, the amount of cortisol after treatment (for example, immediately after treatment) of the same subject.
  • the reference value can be obtained using, for example, a sample isolated from a healthy person and / or a related disease patient (hereinafter also referred to as “reference sample”).
  • reference sample a sample isolated from a healthy person and / or a related disease patient
  • the reference value may be measured simultaneously with the sample of the subject or may be measured in advance.
  • a method for evaluating the risk of affliction of a subject's related disease is not particularly limited, and can be appropriately determined according to the type of the related disease and the reference value.
  • a disease caused by an increase in cortisol when the amount of cortisol in the subject sample is significantly higher than the amount of cortisol in the reference sample of the healthy subject, in the reference sample of the related disease patient If the amount of cortisol is the same (no significant difference) and / or significantly higher than the amount of cortisol in the reference sample of the related disease patient, the subject is at risk of developing the related disease. Can be evaluated as being at or high risk.
  • the amount of cortisol in the subject sample is the same as the amount of cortisol in the healthy subject reference sample (when there is no significant difference), it is significantly lower than the amount of cortisol in the healthy subject reference sample. If and / or significantly lower than the amount of cortisol in the reference sample of the patient with the relevant disease, the subject can be assessed as having no or low risk of suffering from the relevant disease.
  • the subject sample is significantly lower than the amount of cortisol in the healthy subject reference sample, If the amount is the same (no significant difference) and / or significantly lower than the amount of cortisol in the reference sample of the related disease patient, the subject is at risk of suffering from the related disease or It can be evaluated that the risk is high.
  • the amount of cortisol in the subject sample is the same as the amount of cortisol in the reference sample of the healthy subject (when there is no significant difference)
  • the amount of cortisol in the reference sample of the healthy subject is significantly higher. If and / or significantly higher than the amount of cortisol in the reference sample of the patient with the relevant disease, the subject can be assessed as having no or low risk of suffering from the relevant disease.
  • Example 1 The analytical sensor of the present invention was prepared, and cortisol was analyzed by fluorescence detection using the sensor.
  • the polynucleotides of SEQ ID NOs: 1 to 555 shown in Tables 1 to 4 were synthesized and used as the sensor for analysis.
  • the polynucleotides of SEQ ID NOs: 300 to 555 are a combination of (1) to (128) shown in Table 5 above, combining the first strand (ss1) and the second strand (ss2) to form a double strand Used as a nucleic acid sensor.
  • a sensor reagent including the analytical sensor was prepared by the following procedure.
  • a sensor reagent containing the single-stranded nucleic acid sensor was prepared by the following procedure. First, the polynucleotides of SEQ ID NOs: 1 to 299 were each dissolved in distilled water so that the final concentration of the polynucleotide was 10 ⁇ mol / L to prepare a polynucleotide solution. Next, 2 ⁇ L of the polynucleotide solution was added to 25 ⁇ L of Buffer A to prepare a diluted polynucleotide solution. The composition of the buffer A was 100 mmol / L Tris-HCl (pH 7.4) buffer containing 0.05% Triton (trademark) X-100.
  • a sensor reagent including the double-stranded nucleic acid sensor was prepared by the following procedure.
  • the polynucleotides of SEQ ID NOs: 300 to 427 were each dissolved in distilled water so as to have a final concentration of 10 ⁇ mol / L to prepare a first strand solution.
  • the polynucleotides of SEQ ID NOs: 428 to 555 were dissolved in distilled water so as to have a final concentration of 10 ⁇ mol / L, respectively, thereby preparing second strand solutions.
  • 2 ⁇ L of the first strand solution and 4 ⁇ L of the second strand solution were added to 25 ⁇ L of Buffer A to prepare a diluted polynucleotide solution. Except for this point, it was prepared in the same manner as the sensor reagent containing the single-stranded nucleic acid sensor.
  • Example 2 The analytical sensor of the present invention was prepared, and cortisol at different concentrations was analyzed by fluorescence detection using the sensor.
  • the sensor reagent As the sensor reagent, the sensor reagent containing the polynucleotides of SEQ ID NOs: 142 and 288, respectively, was used, except that the final concentration of cortisol in the reaction solution was a predetermined concentration (0, 100, 300, or 1000 ⁇ mol / L). The fluorescence intensity was measured in the same manner as in Example 1. Further, as a control, the fluorescence intensity was measured in the same manner except that the sensor reagent was not added.
  • FIG. 1 is a graph showing the fluorescence intensity of the analytical sensor.
  • the horizontal axis indicates the concentration of cortisol
  • the vertical axis indicates the fluorescence intensity.
  • all the sensors showed high fluorescence intensity with respect to the control.
  • the fluorescence intensity increased as the concentration of cortisol increased. From these results, it was found that the concentration of cortisol in the sample can be analyzed by measuring the fluorescence intensity using the analytical sensor of the present invention.
  • Example 3 The analytical sensor of the present invention was prepared, and cortisol was analyzed by color detection using the sensor.
  • the polynucleotide of SEQ ID NO: 142 was dissolved in distilled water so that the final concentration of the polynucleotide was 100 ⁇ mol / L to prepare a polynucleotide solution.
  • 1 ⁇ L of the polynucleotide solution was added to 50 ⁇ L of the buffer A to prepare a diluted polynucleotide solution.
  • the prepared solution was mixed and then incubated at 95 ° C. for 5 minutes using a heat block. After the incubation, incubation was performed at room temperature (around 25 ° C.), and the temperature of the solution was adjusted to room temperature.
  • the cortisol sample was added to the sensor reagent so that the final concentration of cortisol was a predetermined concentration (0, 111, 333, or 1000 ⁇ mol / L) to prepare a reaction solution. After mixing each reaction solution, it was added to a 96-well plate (Greiner), and 5 ⁇ L of 20 mmol / L ABTS (2,2′-Azinobis (3-ethylbenzothiazolin-6-sulfonic acid)) solution was added to each well. And 1 ⁇ L of a 10 mmol / L aqueous hydrogen peroxide solution were added and mixed. Immediately after the mixing, the absorbance of each reaction solution was measured. For the measurement of absorbance, the measurement apparatus (TECAN infinite M1000 PRO, manufactured by TECAN) was used, and the measurement wavelength was 415 nm. Further, as a control, the absorbance was measured in the same manner except that the sensor sample was not added.
  • FIG. 2 is a graph showing the absorbance of the analytical sensor.
  • the horizontal axis indicates the concentration of cortisol
  • the vertical axis indicates the absorbance.
  • all the sensors showed high absorbance with respect to the control.
  • the absorbance of each sensor increased as the concentration of cortisol increased. From these results, it was found that the cortisol concentration in the sample can be analyzed by measuring the absorbance using the analytical sensor of the present invention.
  • Example 4 The analytical sensor of the present invention was prepared, and cortisol was analyzed by capillary electrophoresis using the sensor.
  • the 5 ′ end of the polynucleotide of SEQ ID NO: 142 was labeled with a fluorescent dye (TYE (trademark) 665, Integrated DNA Technologies, manufactured by MBL).
  • the labeled polynucleotide was dissolved in buffer B so that the final concentration of the polynucleotide was 0.2 ⁇ mol / L, thereby preparing a sensor reagent.
  • the composition of the buffer B was a 40 mmol / L HEPES (pH 7.5) buffer containing 125 mmol / L NaCl, 5 mmol / L KCl, and 1 mmol / L MgCl 2 .
  • the sensor reagent was mixed with an equal amount of sample buffer, incubated at 95 ° C. for 5 minutes, and further incubated on ice for 5 minutes.
  • the composition of the sample buffer was 40 mmol / L HEPES buffer (pH 7.5) containing 20 mmol / L KCl and 0.01% Tween20. After the incubation, the cortisol sample was added to the mixed solution so that the final concentration of cortisol was a predetermined concentration (0, 2, or 5 mmol / L) to prepare a reaction solution.
  • electrophoresis gel (0.6% hydroxypropylmethylcellulose gel, manufactured by SIGMA), measuring chip (i-chip 12, manufactured by Hitachi Chemical) and measuring device (SV12120 Cosmo Eye, Hitachi, respectively) Electrophoresis was performed using a high technology company. In the electrophoresis, the concentration voltage was 600 V, the concentration time was 120 seconds, the separation voltage was 350 V, and the separation time was 240 seconds. Then, with respect to the electrophoresis gel, the fluorescence intensity at each migration distance was measured using the measurement apparatus with an excitation wavelength of 635 nm and an emission wavelength of 660 nm with reference to the migration start point.
  • FIG. 3 is a graph showing the fluorescence intensity at each migration distance of the electrophoresis gel.
  • the horizontal axis represents the migration distance with the migration start point as a reference, and the vertical axis represents the fluorescence intensity.
  • the peak around 400 nm is the detection peak of the complex of the analytical sensor and cortisol
  • the peak around 440 nm is the detection peak of the analytical sensor alone.
  • the concentration of cortisol increased, the fluorescence intensity of the peak around 400 nm increased and the fluorescence intensity of the peak around 440 nm decreased. From these results, it was found that the concentration of cortisol in the sample can be analyzed by capillary electrophoresis using the analytical sensor of the present invention.
  • Example 5 The analytical sensor of the present invention was prepared, and cortisol was analyzed by detecting gold colloid using the sensor.
  • the mixture was incubated at room temperature for 20 minutes.
  • the absorbance at 450 to 650 nm was measured (absorbance before aggregation) using the measurement apparatus (TECAN infinite M1000 PRO, manufactured by TECAN).
  • 1.5 ⁇ L of a 5 mol / L NaCl aqueous solution was added to each well and then stirred for 10 seconds with the measuring device.
  • the mixture was incubated at room temperature for 5 minutes.
  • the absorbance at 450 to 650 nm was measured using the measuring apparatus (absorbance after aggregation).
  • the absorbance before aggregation and the absorbance after aggregation were measured in the same manner except that the sensor reagent was not added. For each wavelength, correction was performed by subtracting the absorbance before aggregation from the absorbance after aggregation, and then the relative value of the absorbance at 650 nm with respect to the absorbance at 520 nm after correction was calculated. Furthermore, the absorbance of the reaction solution having a cortisol concentration of 0 ⁇ mol / L was taken as 1, and the relative value of the absorbance at each concentration was calculated.
  • FIG. 4 is a graph showing relative values of absorbance.
  • the horizontal axis indicates the concentration of cortisol, and the vertical axis indicates the relative value of absorbance.
  • all sensors showed a high relative value of absorbance with respect to the control.
  • the relative value of the absorbance increased in any sensor. From these results, it was found that the cortisol concentration in the sample can be analyzed by detecting the colloidal gold using the analytical sensor of the present invention.
  • Example 6 The analytical sensor of the present invention was confirmed to have low cross-reactivity with melatonin, L-tryptophan, cortisone, norepinephrine, epinephrine, and cholic acid.
  • the analytical sensor includes a combination of the polynucleotide of SEQ ID NO: 421 and the polynucleotide of SEQ ID NO: 549 as the first strand (ss1) and the second strand (ss2), respectively.
  • a double-stranded nucleic acid sensor was used.
  • the 5 ′ end of the polynucleotide of SEQ ID NO: 549 was labeled with the fluorescent dye (TYE TM 665).
  • the labeled polynucleotide was dissolved in distilled water so that the final concentration of the polynucleotide was 100 ⁇ mol / L to prepare a second strand solution.
  • the polynucleotide of SEQ ID NO: 421 was dissolved in distilled water so that the final concentration of the polynucleotide was 100 ⁇ mol / L to prepare a first strand solution. Further, 5 ⁇ L of the first strand solution and the second strand solution were added to 25 ⁇ L of Buffer D to prepare a diluted polynucleotide solution.
  • the composition of the buffer solution D was a 100 mmol / L Tris-HCl (pH 7.4) buffer solution containing 0.1% Triton (trademark) X-100 and 300 mmol / L NaCl.
  • the diluted polynucleotide solution was incubated at 95 ° C. for 5 minutes using a heat block. After the incubation, the solution was incubated at room temperature (around 25 ° C.) for 15 minutes, and the temperature of the solution was adjusted to room temperature (around 25 ° C.).
  • FIG. 5 is a graph showing the degree of fluorescence polarization.
  • the horizontal axis represents the concentration of each sample
  • the vertical axis represents the degree of fluorescence polarization (mP).
  • the degree of fluorescence polarization of the analytical sensor decreased in the cortisol sample depending on the concentration of cortisol. That is, it bound to cortisol.
  • the melatonin sample, the L-tryptophan sample, the cortisone sample, the norepinephrine sample, the epinephrine sample, and the cholic acid sample did not decrease the fluorescence polarization degree depending on the concentration of each sample. .
  • the analytical sensor does not bind or has low binding to these melatonin, L-tryptophan, cortisone, norepinephrine, epinephrine, and cholic acid. From these results, it was found that the analytical sensor of the present invention has low cross-reactivity with melatonin, L-tryptophan, cortisone, norepinephrine, epinephrine, and cholic acid. It was also found that the concentration of cortisol in the sample can be analyzed by fluorescence polarization using the analytical sensor of the present invention.
  • the senor of the present invention can be used in various detection methods.
  • the G-forming region (D) forms a G-quartet structure by cortisol binding to the binding region (A).
  • the G-forming region (D) in which the G-quartet structure is formed is an active type, and for example, it generates a catalytic function or emits fluorescence by forming a complex with porphyrin. For this reason, for example, by detecting the catalytic function or the fluorescence, cortisol can be easily analyzed.
  • the senor for analyzing cortisol according to the present invention has low cross-reactivity to similar compounds such as melatonin, L-tryptophan, cortisone, norepinephrine, epinephrine, and cholic acid.
  • the sensor for cortisol analysis of the present invention can specifically analyze cortisol, for example.
  • the sensor for cortisol analysis of this invention is applicable to the detection method of various cortisol, it can analyze cortisol in various situations. Therefore, it can be said that the sensor for cortisol analysis of the present invention is an extremely useful tool for analyzing cortisol in fields such as the analysis field, the medical field, and the life science field.

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Abstract

La présente invention concerne : un nouveau détecteur d'analyse du cortisol convenant à l'analyse du cortisol ; un procédé d'analyse du cortisol ; un réactif d'évaluation du stress ; un procédé d'évaluation du stress ; un réactif de test destiné à une maladie liée au cortisol ; et un procédé de test destiné au risque de contraction d'une maladie liée au cortisol. La présente invention concerne un détecteur d'analyse du cortisol, ledit détecteur comprenant au moins une molécule d'acide nucléique sélectionnée dans le groupe constitué de (I), (II) et (III) contenant chacun une région de liaison (A) qui se lie à une cible et une région de formation G (D) qui forme une structure G-quartet, est caractérisé en ce que : la cible est le cortisol ; en l'absence de la cible, la formation d'une structure G-quartet est inhibée et ainsi la région de formation G (D) devient inactive ; et en présence de la cible, la région de formation G (D) forme une structure G-quartet et devient active.
PCT/JP2016/071937 2015-12-11 2016-07-26 Détecteur destiné à l'analyse du cortisol, procédé d'analyse du cortisol, réactif d'évaluation du stress, procédé d'évaluation du stress, réactif de test destiné à une maladie liée au cortisol, et procédé de test destiné au risque de contraction d'une maladie liée au cortisol WO2017098746A1 (fr)

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