WO2017098746A1

WO2017098746A1 - Sensor for cortisol analysis, method for cortisol analysis, reagent for stress evaluation, method for stress evaluation, test reagent for cortisol-related disease, and test method for contraction risk of cortisol-related disease

Info

Publication number: WO2017098746A1
Application number: PCT/JP2016/071937
Authority: WO
Inventors: 金子　直人; 宏貴皆川; 穣秋冨; 克紀堀井; 行大白鳥; 巌和賀
Original assignee: Ｎｅｃソリューションイノベータ株式会社
Priority date: 2015-12-11
Filing date: 2016-07-26
Publication date: 2017-06-15
Also published as: JPWO2017098746A1; JP6642859B2

Abstract

Provided are: a novel sensor for cortisol analysis that is usable for analyzing cortisol; a method for cortisol analysis; a reagent for stress evaluation; a method for stress evaluation; a test reagent for a cortisol-related disease; and a test method for the contraction risk of a cortisol-related disease. The sensor for cortisol analysis according to the present invention, said sensor comprising at least one nucleic acid molecule selected from the group consisting of (I), (II) and (III) each containing a binding region (A) that binds to a target and a G-formation region (D) that forms a G-quartet structure, is characterized in that: the target is cortisol; in the absence of the target, the formation of a G-quartet structure is inhibited and thus the G-formation region (D) becomes inactive; and in the presence of the target, the G-formation region (D) forms a G-quartet structure and becomes active.

Description

Sensor for cortisol analysis, cortisol analysis method, stress evaluation reagent, stress evaluation method, test reagent for cortisol-related disease, and method for testing morbidity of cortisol-related disease

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.

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 (Non-Patent Document 1).

Therefore, 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 (hereinafter also referred to as “sensor”) 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. Comprising at least one nucleic acid molecule selected from the group consisting of (II) and (III),
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.
(I) having 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).
(II) having 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) 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). _A double-stranded nucleic acid molecule having a complementary sequence and the stem-forming region (S _A ) having a sequence complementary to the binding region (A).

The cortisol analysis method of the present invention (hereinafter also referred to as “analysis method”) 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 (hereinafter also referred to as “evaluation reagent”) 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.

The cortisol-related disease test reagent of the present invention (hereinafter also referred to as “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 (hereinafter, also referred to as “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 Thus, it is characterized in that it includes a test step for testing the possibility of suffering from a cortisol-related disease.

In the sensor for cortisol analysis of the present invention, 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. In addition, the sensor 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.

<Sensor for cortisol analysis>
As described above, 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.
(I) having 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).
(II) having 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) 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). _A double-stranded nucleic acid molecule having a complementary sequence and the stem-forming region (S _A ) having a sequence complementary to the binding region (A).

Hereinafter, in the present invention, 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. Further, regarding the G-forming region (D), 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).

In the present invention, 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. In the present invention, 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. In addition, in a nucleic acid molecule, 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.

In the sensor of the present invention, 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. For example, ProteON (BioRad) can be used for the analysis.

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. Hereinafter, the description regarding the said cortisol can be used for the said derivative | guide_body.

In the sensor of the present invention, 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 A polynucleotide consisting of a base sequence having 80% or more identity to the base sequence of any one of the above (a1) and binding to the cortisol (a4) from any of the base sequences of the above (a1) Comprising a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to the polynucleotide Polynucleotide that binds to Chizoru

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.

In the polynucleotide (a2), “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. In the present invention, 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).

In the polynucleotide (a3), 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).

In the polynucleotide (a4), 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.

In the above (a4), 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.

In the sensor of the present invention, 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. In the latter case, the binding region (A) may further have, for example, a linker sequence and / or an additional sequence in addition to the polynucleotide.

In the sensor of the present invention, 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.

When 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. In the base sequence of each SEQ ID NO, 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.

In the polynucleotide (a3), when the corresponding conserved base sequence is conserved in the base sequence of each SEQ ID NO, the identity is, for example, 50% or more, 60% or more, 70% or more, 80 %, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.

In the sensor of the present invention, when the G formation region (D) forms a G quartet structure, for example, a catalytic function and fluorescence occur. In 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. For this reason, 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. Thus, in the sensor of the present invention, 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. In the present invention, 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.

First, when the G-forming region (D) forms the G-quartet structure, for example, a complex with porphyrin is formed, whereby the complex emits fluorescence. In this case, the G-quartet nucleic acid is also called a fluorescent nucleic acid.

When the G-forming region (D) is the 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.

Second, when the G-forming region (D) forms the G-quartet structure, for example, a complex with porphyrin is formed, thereby causing the catalytic function of the enzyme. In this case, 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”). Examples of the peroxidase activity 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.

Examples of the DNAzyme 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

When the G-forming region (D) is the catalytic 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. 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.

Note that 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. Examples of the derivatives include substituted porphyrins, metal porphyrins formed with complexes with metal elements, and the like. Examples of 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.

In the G-forming region (D), when the catalytic function is caused, the porphyrin is preferably, for example, the metal porphyrin, and more preferably hemin. In addition, when generating the fluorescence in the G-forming region (D), the porphyrin 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.

When the G-forming region (D) is single-stranded, 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 (d4) and 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. Polynucleotide

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. .

In the above (d2), “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).

In the above (d3), “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.

In (d4), the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (d1). As for “hybridization” and “stringent conditions”, the above description can be used.

In the analytical sensor of the present invention, 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.

When the G-forming region (D) is double-stranded, 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) And 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). The following 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) A polynucleotide consisting of a base sequence having 80% or more identity to any of the base sequences of (f1) (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.

In (e2) above, “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).

In the above (e3), 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.

In the above (e4), the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (e1). As for “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.

In (f2), “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).

In the above (f3), 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.

In the above (f4), the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (f1). As for “hybridization” and “stringent conditions”, the above description can be used.

In the double-stranded G-forming region (D), 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.

Referring to the nucleic acid molecules in the sensor of the present invention, each of (I), (II), and (III) will be described below. In addition, unless otherwise indicated, description of each nucleic acid molecule can each be used.

(1) 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).

In the nucleic acid molecule (I), the G-forming region (D) is, for example, the single-stranded type.

In the nucleic acid molecule (I), for example, based on the following mechanism, 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. Note that the present invention is not limited to this mechanism. In the absence of a target, 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. Further, 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. On the other hand, in the presence of the target, 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. Accordingly, 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). As a result, for example, 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.

For example, 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). Moreover, it is preferable that 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).

In the binding region (A) of the nucleic acid molecule (I), 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 '

In the forms (1) and (2), for example, the formation of the G-quartet structure is turned on and off as follows. In the absence of a target, 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). Then, in the presence of the target, 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).

In (1) and (2), 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).

In the nucleic acid molecule (I), for example, 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.

As a specific example, the following order can be illustrated about the form in which the (1) and (2) further have two intervening linker regions, for example. In the following examples, 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 )”). For example, 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.
_{(1 ') 5'- D-L} 2 -S C -C-L 1 -S D -3'
_{(2 ') 5'- S D -L} 1 -C-S C -L 2 -D -3'

In the forms (1 ′) and (2 ′), for example, the formation of the G-quartet structure is turned on and off as follows. In the absence of the target, for example, 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. Between the two stems, 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). Then, in the presence of the target, 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).

In the nucleic acid molecule (I), 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. For example, 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.

In the nucleic acid molecule (I), 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.

In the nucleic acid molecule (I), 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.

As a specific example, 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 A polynucleotide having a function equivalent to that of (s1), (s3) consisting of a base sequence having 80% or more identity to any of the base sequences of (s1), (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)

In the present invention, “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.

In the polynucleotide of (s2), “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.

In the polynucleotide (s3), “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. .

In the polynucleotide (s4), the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide (s1). As for “hybridization” and “stringent conditions”, the above description can be used.

In the polynucleotides (s2) to (s4), it is preferable that at least one base sequence of the binding region (A) and the G-forming region (D) is conserved in the base sequence of each SEQ ID NO. . 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 (s1), and the corresponding base sequence is the conserved base It can be judged as an array.

In the polynucleotide (s3), when the conserved base sequence is conserved in the base sequence of each SEQ ID NO, 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.

(2) Nucleic acid molecule (II)
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) 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). is there.

In the nucleic acid molecule (II), 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. In the nucleic acid molecule (II), 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 | sequence which forms.

In the nucleic acid molecule (II), for example, based on the following mechanism, 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. As described above, in the nucleic acid molecule (II), 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. Thus, since 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. On the other hand, in the presence of a target, 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.

As described above, 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).

In the nucleic acid molecule (II), 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 | position to 5 'side or 3' side. Hereinafter, unless otherwise specified, for convenience, the first region (D1) is disposed on the 5 ′ side of the coupling region (A), and the second region (D2) is disposed on the 3 ′ side of the coupling region (A). An example is shown.

In the nucleic acid molecule (II), for example, 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.

As described above, 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.

Further, 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. In this case, the nucleic acid molecule in a state where 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 aligned. It can be said that it is a region forming an internal loop in the molecule of (II). Thus, the first linker region (L ₁ ) and the second linker region that are non-complementary between the first region (D1) and the second region (D2) and the binding region (A). By having (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).

In the 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, and 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). ) And 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.

In the arrangement (d1) of the first region (D1) and the arrangement (d2) of the second region (D2), for example, (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.
Condition (3)
(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. . In the condition (1), 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. . Since (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. In the condition (2), 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. For example, 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. In the condition (3), 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. When n3 or m3 is 4, (N) _n3 and (N) _m3 are bases H other than G in the second or third base. (N) _n3 and (N) _m3 with three Gs together with GGG between (N) _n1 and (N) _n2 and GGG between (N) _m1 and (N) _m2 G formation region (D) forming the structure.

N3 and m3 may be any of n3 = m3, n3> m3, and n3 <m3, and preferably n3> m3 and n3 <m3.

Examples of the base H that is a base other than G include A, C, T, and U, and preferably A, C, or T.

Specific examples of the 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.

In the nucleic acid molecule (II), 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.

In the nucleic acid molecule (II), 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) A polynucleotide obtained by replacing the polynucleotide of (e1) with a polynucleotide having an underlined base sequence in any of the base sequences of SEQ ID NOS: 280 to 299 in Table 2, and after the above replacement The explanation can be used. In addition, in the other region, for example, in at least one polynucleotide selected from the group consisting of (f1) to (f4), 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.

As a specific example, 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. A polynucleotide having a function equivalent to that of (t1) (t3) comprising a base sequence having 80% or more identity to any of the base sequences of (t1), A polynucleotide having a function equivalent to that of (t1) (t4) from a nucleotide sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences of (t1) And a polynucleotide having a function equivalent to that of (t1)

In the present invention, “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.

In the polynucleotide (t2), “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.

In the polynucleotide (t3), “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. .

In the polynucleotide (t4), the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide (t1). As for “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. . 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.

In the polynucleotide of (t3), when the conserved base sequence is conserved in the base sequence of each SEQ ID NO, 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.

(3) Nucleic acid molecule (III)
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).

In the nucleic acid molecule (III), the G-forming region (D) is, for example, the single-stranded type.

In the nucleic acid molecule (III), for example, 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. In the absence of a target, 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). As a result, 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. Further, in the molecule, 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. On the other hand, 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. Accordingly, 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). As a result, for example, 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. For this reason, 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.

For example, 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). Moreover, it is preferable that 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).

In the nucleic acid molecule (III), 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 '

In (1), 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. In (2), 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.

In the nucleic acid molecule (III), for example, 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.

As a specific example, for example, the following order can be exemplified for the forms in which (1) and (2) have the intervening linker region in the first chain (ss1) and the second chain (ss2). In the following examples, 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 ). For example, 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.
(1 ') ss1 5'- AL ₁ -D -3'
ss2 3'- S _A -L ₂ -S _D -5 '
(2 ') ss1 5'- DL ₁ -A -3'
_{_{_{ss2 3'- S D -L 2 -S A}}} -5 '

In the forms (1 ′) and (2 ′), for example, the formation of the G-quartet structure is turned on and off as follows. In the absence of the target, for example, 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. Between the two stems, 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). Then, in the presence of the target, 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).

In the nucleic acid molecule (III), 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. For example, 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.

In the nucleic acid molecule (III), 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.

In the nucleic acid molecule (III), 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.

As a specific example, in the nucleic acid molecule (III), 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. A polynucleotide having a function equivalent to that of (u1) (u3) comprising a base sequence having 80% or more identity to any of the base sequences of (u1), A polynucleotide having a function equivalent to that of (u1) (u4) from a nucleotide sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences of (u1) And a polynucleotide having a function equivalent to that of (u1)

In the present invention, “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.

In the polynucleotide (u2), “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.

In the polynucleotide (u3), “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. .

In the polynucleotide (u4), the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide (u1). As for “hybridization” and “stringent conditions”, the above description can be used.

In the polynucleotides (u2) to (u4), it is preferable that at least one base sequence of the binding region (A) and the G-forming region (D) is conserved in the base sequence of each SEQ ID NO. . 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 (u1), and the corresponding base sequence is the conserved base. It can be judged as an array.

In the polynucleotide (u3), when the conserved base sequence is conserved in the base sequence of each SEQ ID NO, 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.

In the nucleic acid molecule (III), 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 (v1) (v3) consisting of a base sequence having 80% or more identity to any of the base sequences of (v1), (V4) 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

In the present invention, “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.

In the polynucleotide (v2), “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.

In the polynucleotide (v3), “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. .

In the polynucleotide (v4), the “hybridizing polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide (v1). As for “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. In the first strand (ss1), for example, in at least one polynucleotide selected from the group consisting of (u1) to (u4), 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. In the second strand (ss2), for example, in at least one polynucleotide selected from the group consisting of (v1) to (v4), 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.

In the nucleic acid molecule (III), 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. For this reason, when the nucleic acid molecule (III) includes the third strand (ss3), 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. Hereinafter, 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).

In the third strand (ss3), 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 | strand (ss1) which becomes a different 1st chain | strand (ss1). The following order can be illustrated as a specific example.
(3) ss1 5'- AD-3 '
ss3 3'- S ' _D -S' _A -5 '
(4) ss1 5'- DA-3 '
_{_{ss3 3'- S 'A -S' D}} -5 '

In (3), 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). In (4), 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. For the direct and indirect connection, 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.

As a specific example, the following order can be illustrated about the form in which the (3) and (4) have the intervening linker region in the first chain (ss1) and the third chain (ss3), for example. In the following examples, 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.
(3 ') ss1 5'- AL ₁ -D -3'
_{_{ss3 3'- S 'D -L 3 -S}} ' A -5 '
(4 ') ss1 5'- DL ₁ -A -3'
ss3 3'- S ' _A -L ₃ -S' _D -5 '

In the third strand (ss3), 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. For example, 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.

When the sensor of the present invention includes the nucleic acid complex (III), the number of the first strand (ss1) and the second strand (ss2) included in the nucleic acid complex (III) is not particularly limited.

In the nucleic acid molecule (III), for example, the first strand (ss1) and the second strand (ss2) may be linked directly or indirectly. When the first strand (ss1) and the second strand (ss2) are linked, 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. It means that the “terminal” and the 5 ′ terminal of the other region are linked via an intervening linker region (L ₁ ), specifically, the 3 ′ end of one region and the intervening linker region This means that the 5 ′ end is directly bonded, and the 3 ′ end of the intervening linker region and the 5 ′ end of the other region are directly bonded. 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.

When the first strand (ss1) and the second strand (ss2) are linked, 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 '

In (5) and (7), 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). In (6) and (8), 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).

In the case where the first strand (ss1) and the second strand (ss2) are linked, 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.

As a specific example, the following order can be exemplified for the forms (5) to (8) having the intervening linker region in the first region and the second region. In the following examples, 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 ). When the first strand (ss1) and the second strand (ss2) are linked, 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.
_{(5 ') 5'- A-L} 2 -D-S D -L 3 -S A -3'
(6 ′) 5′-S _D -L ₃ -S _A -AL ₂ -D -3 '
(7 ′) 5′-AL ₂ -DL ₁ -S _D -L ₃ -S _A -3 ′
(8 ′) 5′-S _D -L ₃ -S _A -L ₁ -AL ₂ -D -3 '

In the forms (5 ′) to (8 ′), for example, the formation of the G-quartet structure is turned on and off as follows. In the absence of the target, for example, 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. Between the two stems, 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). Then, in the presence of the target, 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).

When the first strand (ss1) and the second strand (ss2) are linked, the length of the stem-forming sequence (S _A ) and the stem-forming sequence (S _D ) in the nucleic acid molecule (III) The length 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. Specifically, 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. Examples of the natural base include A, C, G, T, U, and modified bases thereof. Examples of the modification include methylation, fluorination, amination, and thiolation. Examples of 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.

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). As described above, the linker region (L ) Are preferably immobilized at the 3 ′ end of the linker region (L).

In the sensor of the present invention, for example, 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. Specific examples of the linker 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.

In the analytical sensor of the present invention, 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

In the present invention, “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.

In the sensor of the present invention, 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. In the analytical sensor of the present invention, since 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. When the analytical sensor of the present invention has 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. For the porphyrin, for example, the above description can be used.

In addition, the sensor 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. When utilizing the catalytic function of the G-forming region (D), 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.

In the sensor of the present invention, for example, 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. Examples of 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. Examples of the dye include Alexa dyes such as Alexa (registered trademark) 488 and Alexa (registered trademark) 647. Examples of 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.

According to the sensor of the present invention, as described later, for example, cortisol in a sample can be detected.

<Method for analyzing cortisol>
As described above, 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.

According to the analysis method of the present invention, 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.

In the present invention, the sample is not particularly limited. Examples of the sample include a biological sample. Examples of 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. For example, the sample is preferably a liquid sample because it is easy to contact with the nucleic acid molecule and is easy to handle. In the case of the solid sample, for example, 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.

In the contact step, 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.

In the contact step, 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., and the contact time is, for example, 10 to 120 minutes or 30 to 60 minutes.

In the contacting step, the sensor 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.

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. By detecting the presence or absence of binding between the two, for example, the presence or absence of cortisol in the sample can be analyzed (qualitative), and by detecting the degree of binding (binding amount) between the two, for example, the sample The amount of cortisol can be analyzed (quantified). In the former case, the analysis method of the present invention can also be called a detection method, for example.

If 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. For example, 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.

<Analysis reagents and analysis kits>
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. Examples of 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. For 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. Examples of the component include the carrier, the reagent, a buffer solution, and instructions for use.

For example, 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.

<Stress evaluation reagent and evaluation kit>
As described above, 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. For the evaluation reagent and evaluation 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 evaluation method of the present invention to be described later can be cited.

<Stress evaluation method>
As described above, 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.

For the evaluation method of the present invention, for example, the description of the sensor of the present invention and the analysis method of the present invention can be cited. In the evaluation method of the present invention, 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). For example, 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.

In the acquisition step, 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. As a specific example, when 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. Further, 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.

<Test reagents and test kits for cortisol-related diseases>
As described above, 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. For the 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.

<Method for testing morbidity of cortisol-related diseases>
As described above, the method for testing the morbidity of a cortisol-related disease according to the present invention 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. By detecting the cortisol in the sample by binding the binding region (A) in the analytical sensor, and comparing the amount of the cortisol in the detection step with a reference value, It is characterized by including a test process for testing the possibility of morbidity. 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.

For the 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. In the test method of the present invention, the description of the analysis method of the present invention can be used for the contact step and the detection step.

According to the test method of the present invention, 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. As specific examples, 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. Examples of 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. In the case of prognostic evaluation, 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”). In the case of prognostic evaluation, for example, a reference sample isolated from the same subject after treatment may be used. For example, the reference value may be measured simultaneously with the sample of the subject or may be measured in advance.

In the test process, 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. As a specific example, in the case of 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. In addition, when 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. On the other hand, in the case of a disease caused by a decrease in cortisol, if the amount of cortisol in 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. In addition, when 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.

Next, examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercially available reagents were used based on their protocol unless otherwise indicated.

[Example 1]
The analytical sensor of the present invention was prepared, and cortisol was analyzed by fluorescence detection using the sensor.

(1) Sensor for analysis 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. Specifically, 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 μmol / L to prepare a polynucleotide solution. Next, 2 μL of the polynucleotide solution was added to 25 μL 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.

Next, 1.5 μL of a 5 mol / L NaCl aqueous solution was added to the diluted polynucleotide solution. After mixing the obtained solution, it incubated on 95 degreeC and the conditions for 5 minutes using the 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. Furthermore, 25 μL of distilled water, 1 μL of 10 μmol / L NMM aqueous solution and 1 μL of 1 mol / L KCl aqueous solution were added and mixed to prepare a sensor reagent.

Next, a sensor reagent including the double-stranded nucleic acid sensor was prepared by the following procedure. First, the polynucleotides of SEQ ID NOs: 300 to 427 were each dissolved in distilled water so as to have a final concentration of 10 μmol / L to prepare a first strand solution. Further, the polynucleotides of SEQ ID NOs: 428 to 555 were dissolved in distilled water so as to have a final concentration of 10 μmol / L, respectively, thereby preparing second strand solutions. Next, 2 μL of the first strand solution and 4 μL of the second strand solution were added to 25 μL 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.

(2) Sample A cortisol sample dissolved in DMSO was prepared so as to be 100 mmol / L cortisol, and used for the following analysis.

(3) Fluorescence analysis The cortisol sample was added to each sensor reagent so that the final concentration of cortisol was 1 mmol / L to prepare a reaction solution. After mixing each reaction solution, it was added to a 384-well black flat plate (Greiner), and the fluorescence intensity of each reaction solution was measured. The fluorescence intensity was measured using a measuring device (TECAN infinite M1000 PRO, manufactured by TECAN), with an excitation wavelength of 399 nm and an emission wavelength of 605 nm. Further, as a control, the fluorescence intensity was measured in the same manner except that the cortisol sample was not added. And about each analytical sensor, S / N ratio which is a ratio of the fluorescence intensity of the reaction liquid without the sample and the reaction liquid with the sample was obtained.

The results are shown in Tables 7A to C below. In Tables 7A to 7C below, single-stranded sensors are indicated by SEQ ID NOs of polynucleotides included in each sensor, and double-stranded nucleic acid sensors are indicated by SEQ ID NOs of the first strand (ss1) included in each sensor. As shown in Table 7 below, it was found that each sensor had an S / N ratio of 1 or more, and cortisol could be analyzed.

[Example 2]
The analytical sensor of the present invention was prepared, and cortisol at different concentrations was analyzed by fluorescence detection using the sensor.

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 μmol / 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.

The result is shown in FIG. FIG. 1 is a graph showing the fluorescence intensity of the analytical sensor. In FIG. 1, the horizontal axis indicates the concentration of cortisol, and the vertical axis indicates the fluorescence intensity. As shown in FIG. 1, all the sensors showed high fluorescence intensity with respect to the control. Moreover, in any sensor, 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.

(1) Analytical Sensor The polynucleotide of SEQ ID NO: 142 was dissolved in distilled water so that the final concentration of the polynucleotide was 100 μmol / L to prepare a polynucleotide solution. Next, 1 μL of the polynucleotide solution was added to 50 μL 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. Furthermore, 39 μL of distilled water, 1 μL of a 100 μmol / L hemin aqueous solution and 2 μL of a 1 mol / L KCl aqueous solution were added and mixed to prepare a sensor reagent.

(2) Color Analysis 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 μmol / L) to prepare a reaction solution. After mixing each reaction solution, it was added to a 96-well plate (Greiner), and 5 μL of 20 mmol / L ABTS (2,2′-Azinobis (3-ethylbenzothiazolin-6-sulfonic acid)) solution was added to each well. And 1 μL 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.

This result is shown in FIG. FIG. 2 is a graph showing the absorbance of the analytical sensor. In FIG. 2, the horizontal axis indicates the concentration of cortisol, and the vertical axis indicates the absorbance. As shown in FIG. 2, all the sensors showed high absorbance with respect to the control. In addition, 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.

(1) Sensor for analysis 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 μmol / 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 .

(2) Capillary electrophoresis 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. For 10 μL of each 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.

This result is shown in FIG. FIG. 3 is a graph showing the fluorescence intensity at each migration distance of the electrophoresis gel. In FIG. 3, the horizontal axis represents the migration distance with the migration start point as a reference, and the vertical axis represents the fluorescence intensity. In FIG. 3, the peak around 400 nm is the detection peak of the complex of the analytical sensor and cortisol, and the peak around 440 nm is the detection peak of the analytical sensor alone. As shown in FIG. 3, as 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.

(1) Sensor for analysis The polynucleotides of SEQ ID NOs: 28 and 142 were each dissolved in distilled water so that the final concentration of the polynucleotide was 2 μmol / L to prepare a sensor reagent.

(2) Detection of gold colloid 5 μL of the sensor reagent and 45 μL of 10 nm colloidal gold dispersion (catalog number: G1527-25ML) so that the final concentration of the polynucleotide is 0.2 μmol / L. Was added to a 96 Well U bottom plate (manufactured by Greiner) and stirred using a plate shaker at room temperature, 10 seconds, and 1000 rpm. After the stirring, the mixture was incubated at room temperature for 1 hour. Further, the cortisol sample was added to each well so that the final concentration of cortisol was a predetermined concentration (0, 4, 40, or 400 μmol / L) to prepare a reaction solution. The reaction solution was stirred using a plate shaker under the conditions of room temperature, 10 seconds, and 1000 rpm.

After the stirring, the mixture was incubated at room temperature for 20 minutes. Next, with respect to each well of the plate, the absorbance at 450 to 650 nm was measured (absorbance before aggregation) using the measurement apparatus (TECAN infinite M1000 PRO, manufactured by TECAN). Further, 1.5 μL of a 5 mol / L NaCl aqueous solution was added to each well and then stirred for 10 seconds with the measuring device. After the stirring, the mixture was incubated at room temperature for 5 minutes. Then, for each well of the plate, the absorbance at 450 to 650 nm was measured using the measuring apparatus (absorbance after aggregation). For the control, 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 μmol / L was taken as 1, and the relative value of the absorbance at each concentration was calculated.

This result is shown in FIG. FIG. 4 is a graph showing relative values of absorbance. In FIG. 4, the horizontal axis indicates the concentration of cortisol, and the vertical axis indicates the relative value of absorbance. As shown in FIG. 4, all sensors showed a high relative value of absorbance with respect to the control. Moreover, as for the concentration of cortisol, 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.

(1) Analytical sensor 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.

First, the 5 ′ end of the polynucleotide of SEQ ID NO: 549 was labeled with the fluorescent dye (TYE ™ 665). The labeled polynucleotide was dissolved in distilled water so that the final concentration of the polynucleotide was 100 μmol / L to prepare a second strand solution.

Next, the polynucleotide of SEQ ID NO: 421 was dissolved in distilled water so that the final concentration of the polynucleotide was 100 μmol / L to prepare a first strand solution. Further, 5 μL of the first strand solution and the second strand solution were added to 25 μL 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. Then, 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.).

(2) Sample Cortisol, melatonin, L- so that when a reaction solution described later is prepared, the final concentration of each compound in the reaction solution is a predetermined concentration (0, 100, 500, or 1000 μmol / L). Tryptophan, cortisone, DL-norepinephrine hydrochloride, (±) -epinephrine hydrochloride, and cholic acid are each dissolved in DMSO, cortisol sample, melatonin sample, L-tryptophan sample, cortisone sample, norepinephrine sample, epinephrine sample, and A cholic acid sample was prepared.

(3) Fluorescence Polarization Method A reaction solution containing 35 μL of the diluted polynucleotide, 1 μL of 1 mol / L KCl aqueous solution, 13.5 μL of distilled water, and 0.5 μL of the sample was prepared for each sample. After stirring the reaction solution, each was added to a 384 Well flat bottom black plate (Greiner), and the degree of fluorescence polarization at an excitation wavelength of 635 nm and a fluorescence wavelength of 670 nm was measured using the measuring device. The fluorescence polarization degree was calculated as a relative value when the control polarization degree of the sample to which the polynucleotide of SEQ ID NO: 421 and the sample were not added was 20 mP.

This result is shown in FIG. FIG. 5 is a graph showing the degree of fluorescence polarization. In FIG. 5, the horizontal axis represents the concentration of each sample, and the vertical axis represents the degree of fluorescence polarization (mP). As shown in FIG. 5, 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. In contrast, in the analytical sensor, 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. . That is, it was found that 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.

From the above, it was found that the sensor of the present invention can be used in various detection methods.

As mentioned above, although this invention was demonstrated with reference to embodiment and an Example, this invention is not limited to the said embodiment and Example. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

This application claims priority based on Japanese Patent Application No. 2015-241924 filed on Dec. 11, 2015, the entire disclosure of which is incorporated herein.

In the sensor for cortisol analysis of the present invention, 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. In addition, the sensor 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. And since 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.

Claims

At least one nucleic acid molecule selected from the group consisting of the following (I), (II), and (III), comprising a binding region (A) that binds to a target and a G-forming region (D) that forms a G-quartet structure Including
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) forms a G-quartet structure to become an active type, and a sensor for cortisol analysis.
(I) having 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).
(II) having 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) 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). A double-stranded nucleic acid molecule having a complementary sequence and the stem-forming region (S A ) having a sequence complementary to the binding region (A).
The sensor for cortisol analysis according to claim 1, wherein in the presence of the target, the G-forming region (D) forms a G-quartet structure to form a complex with porphyrin, whereby the complex generates fluorescence.
The sensor for cortisol analysis according to claim 1, wherein in the presence of the target, the G-forming region (D) forms a G-quartet structure to form a complex with porphyrin, thereby generating a catalytic function.
In the nucleic acid molecule (I), the G-forming region (D), the stem-forming region (S D ), the intermediate region (C) and the stem-forming region (S C ) are the following (1) or (2) The sensor for cortisol analysis according to any one of claims 1 to 3, wherein the sensors are connected in the following order.
(1) Order of the G formation region (D), the stem formation region (S C ), the intermediate region (C), and the stem formation region (S D ) (2) The stem formation region (S D ), Order of the intermediate region (C), the stem formation region (S C ), and the G formation region (D)
The nucleic acid molecule (I) is further
Including an internal region (I D ) and an internal region (I A ),
An internal region (I D ) is disposed between the stem formation region (S C ) and the G formation region (D),
An internal region (I C ) is disposed between the stem formation region (S D ) and the intermediate region (C),
The sensor for cortisol analysis according to any one of claims 1 to 4, wherein the internal region (I D ) and the internal region (I C ) are non-complementary to each other.
In the nucleic acid molecule (I), the binding region (A) includes a polynucleotide having a base sequence surrounded by a square in any one of the base sequences of SEQ ID NOS: 1 to 279 of Tables 1A to G and H below. Item 6. The sensor for cortisol analysis according to any one of Items 1 to 5.
The nucleic acid molecule (I), wherein the G-forming region (D) comprises 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. The sensor for cortisol analysis according to any one of 1 to 6.
The sensor for cortisol analysis according to any one of claims 1 to 7, wherein the nucleic acid molecule (I) comprises a polynucleotide comprising at least one base sequence selected from the group consisting of SEQ ID NOs: 1 to 279.
The nucleic acid molecule (II), wherein the binding region (A) comprises a polynucleotide comprising a base sequence surrounded by a square in any one of the base sequences of SEQ ID NOS: 280 to 299 in Table 2 below. The sensor for cortisol analysis as described in any one of these.
In the nucleic acid molecule (II), one of the first region (D1) and the second region (D2) is composed of an underlined base sequence in any one of the base sequences of SEQ ID NOs: 280 to 299 in Table 2. Comprising a polynucleotide;
The sensor for cortisol analysis according to any one of claims 1 to 3 and 9, wherein the other region comprises a polynucleotide comprising an underlined base sequence surrounded by parentheses in the base sequence of the same SEQ ID NO.
The cortisol according to any one of claims 1 to 3, 9, and 10, wherein the nucleic acid molecule (II) comprises a polynucleotide comprising at least one base sequence selected from the group consisting of SEQ ID NOs: 280 to 299. Sensor for analysis.
In the nucleic acid molecule (III), the binding region (A) of the first strand (ss1) is a base sequence surrounded by a square in any one of the base sequences of SEQ ID NOS: 300 to 427 in Tables 3A to C and D below. The sensor for cortisol analysis as described in any one of Claim 1 to 3 containing the polynucleotide which consists of.
In the nucleic acid molecule (III), the G-forming region (D) of the first strand (ss1) is an underlined base in any one of the nucleotide sequences of SEQ ID NOS: 300 to 427 in Tables 3A to C and D The sensor for cortisol analysis according to any one of claims 1 to 3 and 12, comprising a polynucleotide comprising a sequence.
The nucleic acid molecule (III), wherein the first strand (ss1) comprises a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOs: 300 to 427 in Tables 3A to C and D. The sensor for cortisol analysis according to any one of 14 and 13.
The nucleic acid molecule (III), wherein the second strand (ss2) comprises a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 428 to 555 in Tables 4A and B below: The sensor for cortisol analysis as described in any one of these.
The combination of the first strand (ss1) and the second strand (ss2) of the nucleic acid molecule (III) is at least one selected from the group consisting of (1) to (127) and (128) in Table 5 below A combination of
The said 1st strand (ss1) and the said 2nd strand (ss2) respectively contain the polynucleotide which consists of a base sequence of the sequence number corresponding to the said combination, Each one of Claim 1 to 3 and 12 to 15 The sensor for cortisol analysis described in 1.
Furthermore, a linker region (L) is included,
The sensor for cortisol analysis according to any one of claims 1 to 16, wherein the linker region (L) is linked to an end of the nucleic acid molecule.
The sensor for cortisol analysis according to claim 17, wherein the linker region (L) is linked to the 3 'end of the nucleic acid molecule.
The sensor for cortisol analysis according to claim 18, wherein the 3 'end of the linker region (L) is immobilized.
And further comprising a carrier,
The sensor for cortisol analysis according to any one of claims 1 to 19, wherein the nucleic acid molecule is immobilized on the carrier.
The sensor for cortisol analysis according to claim 20, further comprising a reagent that reacts with the G-forming region (D).
The sensor for cortisol analysis according to claim 21, wherein the reagent contains porphyrin that forms a complex with the G-forming region (D) having a G-quartet structure.
The sensor for cortisol analysis according to claim 22, wherein the porphyrin is at least one porphyrin selected from the group consisting of N-methylmesoporphyrin, Zn-DIGP, ZnPP9 and TMPyP.
The sensor for cortisol analysis according to any one of claims 21 to 23, wherein the reagent contains a substrate for the catalytic function of the G-forming region (D) having a G-quartet structure.
The sensor for cortisol analysis according to any one of claims 1 to 24, wherein the polynucleotide is DNA.
The sensor for cortisol analysis according to any one of claims 1 to 25, wherein the polynucleotide comprises a modified base.
A contact step of bringing a sample into contact with the sensor for cortisol analysis according to any one of claims 1 to 26, and binding of cortisol in the sample to the binding region (A) in the sensor for cortisol analysis And a detection step of detecting cortisol in the sample.
28. The cortisol analysis method according to claim 27, wherein the sample is a biological sample.
29. The cortisol analysis method according to claim 27 or 28, wherein the sample is at least one sample selected from the group consisting of blood, serum, plasma, interstitial fluid, urine, saliva, sweat, tears, and runny nose.
The cortisol according to any one of claims 27 to 29, wherein in the detection step, the cortisol is detected by detecting fluorescence due to a complex of the binding region (A) and the porphyrin in the presence of porphyrin. Analysis method.
The cortisol analysis method according to claim 30, wherein, in the detection step, the detection of the fluorescence is a measurement of fluorescence intensity.
30. The cortisol analysis method according to any one of claims 27 to 29, wherein in the detection step, the cortisol is detected by detecting a catalytic function of the G formation region (D) in the sensor for cortisol analysis.
The cortisol analysis method according to claim 32, wherein in the detection step, the catalytic function is detected in the presence of a substrate for the catalytic function of the G-forming region (D).
A stress evaluation reagent comprising the sensor for cortisol analysis according to any one of claims 1 to 26.
The stress evaluation reagent according to claim 34, further comprising a reagent that reacts with the G-forming region (D) of the sensor for cortisol analysis.
The stress evaluation reagent according to claim 35, wherein the reagent contains porphyrin forming a complex with the G-forming region (D) having a G-quartet structure.
The stress evaluation reagent according to claim 36, wherein the porphyrin is at least one porphyrin selected from the group consisting of N-methylmesoporphyrin, Zn-DIGP, ZnPP9 and TMPyP.
The stress evaluation reagent according to any one of claims 35 to 37, wherein the reagent contains a substrate for the catalytic function of the G-forming region (D) having a G-quartet structure.
A contact step of bringing a sample of the subject into contact with the sensor for cortisol analysis according to any one of claims 1 to 26;
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 A stress evaluation method comprising an acquisition step of acquiring information related to stress.
A test reagent for cortisol-related diseases, comprising the sensor for analyzing cortisol according to any one of claims 1 to 26.
A contact step of bringing a sample of the subject into contact with the sensor for cortisol analysis according to any one of claims 1 to 26;
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 A test method for testing the possibility of cortisol-related disease, comprising a test step of testing the possibility of suffering from a cortisol-related disease.