# Patent

## Title
Device and method for analyzing target

## Abstract

The present invention provides a novel sensor for detecting a target. The nucleic acid sensor of the present invention includes a nucleic acid element that includes a catalyst nucleic acid molecule (D) that exerts a catalytic function and a binding nucleic acid molecule (A) that binds to a target. The nucleic acid element is a double-stranded nucleic acid element including a first strand and a second strand. The first strand (ss1) includes the binding nucleic acid molecule (A), a loop-forming sequence (L1), and the catalyst nucleic acid molecule (D) linked in this order. The second strand (ss2) includes a stem-forming sequence (SA), a loop-forming sequence (L2), and a stem-forming sequence (SD) linked in this order. In this nucleic acid element, in the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited by stem formation in each of the stem-forming sequences (SA) and (SD), and in the presence of ATP/target, the stem formation is released by a binding of the binding nucleic acid molecule (A) with the target, and the catalytic function is exerted.

## Background
TECHNICAL FIELD
The present invention relates to a nucleic acid sensor, a device, and a method for analyzing a target.
BACKGROUND ART
In recent years, an increase in food poisoning caused by infection with microorganisms such as Escherichia coli and Salmonella enterica has been a problem. A major cause of this problem is a washing failure. Therefore, it is necessary to conduct a cleanliness inspection in a food-processing factory.
As such inspection, a method in which cleanliness is analyzed by measuring ATP (adenosine triphosphate) or AMP (adenosine monophosphate) has been put into practical use in recent years. ATP and AMP are known as necessary components for energy metabolism in various organisms. Therefore, it is considered that the amount of ATP or AMP has a correlation to the amount derived from the microorganisms and food present therein. Thus, by measuring ATP or AMP in an objective region, the extent of microorganisms and food residues remaining in the region can be analyzed. Accordingly, cleanliness can be evaluated.
As a tool for detecting AMP, for example, a nucleic acid element obtained by linking a DNA aptamer that specifically binds to AMP with DNA (hereinafter merely referred to as DNAzyme) that exerts peroxidase activity has been reported (Non-Patent Document 1). The structure of this nucleic acid element is controlled so that a catalytic ability of DNAzyme is inhibited by self-association of an aptamer with the DNAzyme in the absence of AMP, and the self-association is released by a binding of AMP to the aptamer in the presence of AMP, thereby exerting the catalytic ability of the DNAzyme. Therefore, for example, when AMP is present, a peroxidase reaction occurs by DNAzyme exerting a catalytic ability. Thus, the amount of AMP can be measured indirectly by meaning the reaction. On the other hand, when AMP is not present, the catalytic ability of DNAzyme is not exerted. Thus, the peroxidase reaction does not occur. However, such nucleic acid element is required to further improve the sensitivity, the ease of handling, and the like in order to put the nucleic acid element into practical use. Such requirement is applied not only to AMP but also ATP and the other targets.
PRIOR ART DOCUMENT
Non-Patent Document


    Non-Patent Document 1: Carsten Teller et. al., Anal. Chem. 2009, 81, 9114-9119


SUMMARY OF INVENTION
Hence, the present invention is intended to provide a novel nucleic acid sensor for detecting a target.
A nucleic acid sensor for analyzing a target of the present invention is a nucleic acid sensor for analyzing a target, including the following nucleic acid element (I), (II), (II′), or (III) that includes a catalyst nucleic acid molecule (D) that exerts a catalytic function and a binding nucleic acid molecule (A) that binds to a target,
(I) a double-stranded nucleic acid element including a first strand and a second strand, the first strand (ss1) including the binding nucleic acid molecule (A), a loop-forming sequence (L1), and the catalyst nucleic acid molecule (D) linked in this order, the second strand (ss2) including a stem-forming sequence (SA), a loop-forming sequence (L2), and a stem-forming sequence (SD) linked in this order, wherein a terminal region of the binding nucleic acid molecule (A) in the first strand (ss1) on the loop-forming sequence (L1) side is complementary to the stem-forming sequence (SA) in the second strand (ss2), a terminal region of the catalyst nucleic acid molecule (D) in the first strand (ss1) on the loop-forming sequence (L1) side is complementary to the stem-forming sequence (SD) in the second strand (ss2), the loop-forming sequence (L1) in the first strand (ss1) is non-complementary to the loop-forming sequence (L2) in the second strand (ss2), and in the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited by stem formation in each of the stem-forming sequences (SA) and (SD), and in the presence of a target, the stem formation in each of the stem-forming sequences (SA) and (SD) is released by a binding of the target with the binding nucleic acid molecule (A), and the catalytic function of the catalyst nucleic acid molecule (D) is exerted,

(II) a single-stranded nucleic acid element including the binding nucleic acid molecule (A), a loop-forming sequence (L1), a stem-forming sequence (SD), the catalyst nucleic acid molecule (D), a loop-forming sequence (L2), and a stem-forming sequence (SA) linked in this order, wherein a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side is complementary to the stem-forming sequence (SA), a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side is complementary to the stem-forming sequence (SD), the loop-forming sequence (L1) is non-complementary to the loop-forming sequence (L2), and in the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited by stem formation in each of the stem-forming sequences (SA) and (SD), and in the presence of a target, the stem formation in each of the stem-forming sequences (SA) and (SD) is released by a binding of the target with the binding nucleic acid molecule (A), and the catalytic function of the catalyst nucleic acid molecule (D) is exerted, (II′) a single-stranded nucleic acid element including the catalyst nucleic acid molecule (D), a loop-forming sequence (L2), a stem-forming sequence (SA), the binding nucleic acid molecule (A), a loop-forming sequence (L1), and a stem-forming sequence (SD) linked in this order, wherein a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side is complementary to the stem-forming sequence (SD), a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side is complementary to the stem-forming sequence (SA), the loop-forming sequence (L1) is non-complementary to the loop-forming sequence (L2), and in the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited by stem formation in each of the stem-forming sequences (SA) and (SD), and in the presence of a target, the stem formation in each of the stem-forming sequences (SA) and (SD) is released by a binding of the target with the binding nucleic acid molecule (A), and the catalytic function of the catalyst nucleic acid molecule (D) is exerted, and

(III) a single-stranded nucleic acid element including the catalyst nucleic acid molecule (D), an intervening sequence (I), and the binding nucleic acid molecule (A) linked in this order, wherein the intervening sequence (I) is non-complementary to the catalyst nucleic acid molecule (D) and the binding nucleic acid molecule (A), and in the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited, and in the presence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is exerted.

The device for analyzing a target of the present invention (hereinafter merely referred to as the “analysis device of the present invention”) is a device for analyzing a target, including a base material; a nucleic acid sensor; and a detection part, wherein the nucleic acid sensor and the detection part are arranged in the base material, the nucleic acid sensor is the nucleic acid sensor according to the present invention, and the detection part is a detection part that detects the catalytic function of the catalyst nucleic acid molecule (D) in the nucleic acid sensor.
The method for analyzing a target of the present invention (hereinafter merely referred to as the “analysis method of the present invention”) is a method for analyzing a target, including: a contact step of causing a sample containing a target to be in contact with the nucleic acid sensor according to the present invention; and a detection step of detecting the catalytic function of the catalyst nucleic acid molecule (D) in the nucleic acid sensor to detect a target in the sample.
The analysis method of the present invention is a method for analyzing a target, including a contact step of causing a sample containing a target to be in contact with the analysis device according to the present invention; and a detection step of detecting the catalytic function of the catalyst nucleic acid molecule (D) in the detection part of the analysis device to detect a target in the sample.
According to the nucleic acid sensor of the present invention, the catalytic function of the catalyst nucleic acid molecule (D) can be switched ON/OFF by a binding/non-binding of the binding nucleic acid molecule (A) with a target. Therefore, the presence or absence of and the amount of a target can be easily detected by detecting the catalytic function of the catalyst nucleic acid molecule. Moreover, the analysis device of the present invention uses the nucleic acid sensor as mentioned above. Therefore, for example, the analysis device can be downsized and formed into a chip, and many specimens can be easily analyzed by the analysis device. Thus, it can be said that the present invention is a really useful technology for researches and inspections in various fields such as clinical medical care, food, and environment, for example. In the present invention, the concept of the “analysis” encompasses quantitative analysis, semi-quantitative analysis, and qualitative analysis, for example.



BRIEF DESCRIPTION OF DRAWINGS
FIGS. 1A and 1B are schematic views showing an example of a nucleic acid element in the nucleic acid sensor of the present invention.
FIGS. 2A and 2B are schematic views showing another example of a nucleic acid element in the nucleic acid sensor of the present invention.
FIGS. 3A and 3B are schematic views showing yet another example of a nucleic acid element in the nucleic acid sensor of the present invention.
FIGS. 4A and 4B are schematic views showing yet another example of a nucleic acid element in the nucleic acid sensor of the present invention.
FIG. 5 is a graph showing measurement results of absorbance in Example 1.
FIG. 6 is a graph showing measurement results of absorbance in Example 2.
FIG. 7 is a graph showing measurement results of absorbance in Example 3.
FIG. 8 is a graph showing measurement results of absorbance in Example 4.



DESCRIPTION OF EMBODIMENTS
(Nucleic Acid Sensor and Analysis Method Using the Same)
The nucleic acid sensor for analyzing a target of the present invention includes, as mentioned above, the nucleic acid element (I), (II), (II′), or (III) that includes a catalyst nucleic acid molecule (D) that exerts a catalytic function and a binding nucleic acid molecule (A) that binds to a target.
The binding nucleic acid molecule (A) is not particularly limited as long as it is a nucleic acid molecule that binds to a target. The binding nucleic acid molecule (A) is, for example, also referred to as an aptamer.
In the present invention, the target is not at all limited, and any target can be selected. According to the target, a binding nucleic acid molecule that binds to the target may be used as the binding nucleic acid molecule in the nucleic acid sensor. Examples of the target include ATP, AMP, microorganisms, virus, food allergens, pesticide, and mycotoxin. Examples of the microorganisms include Salmonella enterica, Listeria monocytogenes, Escherichia coli, and mold. Examples of the virus include norovirus. Specifically, the target may be either one of ATP and AMP or both of them, for example. For example, in the former case, the binding nucleic acid molecule (A) may bind to either ATP or AMP, and in the latter case, the binding nucleic acid molecule (A) may bind to both of them. In the present invention, “binding to ATP or AMP” may encompass, in addition to being capable of binding to ATP or AMP, being capable of binding to any of a fragment of ATP or ATP and a derivative of ATP or AMP, for example.
The binding nucleic acid molecule (A) is, for example, a single strand. The length of the binding nucleic acid molecule (A) is not particularly limited. The lower limit thereof is, for example, 18-mer, preferably 20-mer, more preferably 24-mer, and the upper limit thereof is, for example, 120-mer, preferably 60-mer, more preferably 26-mer.
As the binding nucleic acid molecule (A), a nucleic acid molecule whose target is ATP or AMP (hereinafter referred to as an ATP/AMP binding nucleic acid molecule (A)) is shown below as an example. The ATP/AMP binding nucleic acid molecule (A) can be, for example, an ATP/AMP binding nucleic acid molecule that includes the following polynucleotide (a1), (a2), (a3), or (a4). The ATP/AMP binding nucleic acid molecule (A) may be, for example, a molecule that is composed of or includes the polynucleotide. The ATP/AMP binding nucleic acid molecule (A) including the following polynucleotide (a1), (a2), (a3), or (a3) can also be referred to as, for example, a binding DNA molecule.
(a1) a polynucleotide composed of a base sequence of SEQ ID NO: 1,
SEQ ID NO: 1 CCTGGGGGAGTATTGCGGAGGAAGG
(a2) a polynucleotide that is composed of a base sequence obtained by substitution, deletion, addition and/or insertion of at least one base in the base sequence of the polynucleotide (a1) and binds to ATP or AMP,
(a3) a polynucleotide that is composed of a base sequence with 50% or more identity with the base sequence of the polynucleotide (a1) and is bindable to ATP or AMP, and
(a4) a polynucleotide that is composed of a base sequence complementary to a base sequence that hybridizes to the base sequence of the polynucleotide (a1) under stringent conditions and is bindable to ATP or AMP.
In the polynucleotide (a2), “at least one” is not particularly limited as long as the polynucleotide (a2) binds to ATP or AMP. The number of substituted bases is, in the base sequence of the polynucleotide (a1), for example, from 1 to 5, preferably from 1 to 4, more preferably from 1 to 3, yet more preferably 1 or 2, particularly preferably 1. The number of added or inserted bases is, in the base sequence of the polynucleotide (a1), for example, from 1 to 5, preferably from 1 to 4, more preferably from 1 to 3, yet more preferably 1 or 2, particularly preferably 1. The number of deleted bases is, in the base sequence of the polynucleotide (la), for example, from 1 to 5, preferably from 1 to 4, more preferably from 1 to 3, yet more preferably 2 or 1, particularly preferably 1.
In the polynucleotide (a3), the identity is, to the base sequence of the polynucleotide (a1), for example, 70% or more, preferably 80% or more, more preferably 90% or more, yet more preferably 95% or more, 96% or more, 97% or more, 98% or more, particularly preferably 99% or more. The identity can be calculated using BLAST or the like under default conditions, for example.
In the polynucleotide (a4), “hybridization under stringent conditions” means hybridization under experimental conditions well known to those of ordinary skill in the art, for example. Specifically, the “stringent conditions” refer to conditions under which the base sequence can be identified after conducting hybridization at 60° C. to 68° C. in the presence of 0.7 to 1 mol/l NaCl and then washing at 65° C. to 68° C. using a 0.1- to 2-fold SSC solution, for example. Note here that 1×SSC is composed of 150 mmol/L NaCl and 15 mmol/L sodium citrate.
The ATP/AMP binding nucleic acid molecule (A) is not limited by these examples as long as it is a nucleic acid molecule that binds to ATP or AMP as mentioned above.
The binding nucleic acid molecule (A) is, for example, a molecule including a nucleotide residue and may be a molecule composed of only a nucleotide residue or a molecule including a nucleotide residue. Examples of the nucleotide include ribonucleotide, deoxyribonucleotide, and derivatives thereof. The binding nucleic acid molecule (A) may include, for example, only one kind of ribonucleotide, deoxyribonucleotide, and derivatives thereof, two or more kinds of them, or all of them. Specifically, the nucleic acid molecule may be, for example, DNA including deoxyribonucleotide and/or a derivative thereof, RNA including ribonucleotide and/or a derivative thereof, or chimera (DNA/RNA) including the former and the latter.
The nucleotide may include, for example, either a natural base (non-artificial base) or a non-natural base (artificial base). Examples of the natural base include A, C, G, T, and U and modified bases thereof. Examples of the modification include methylation, fluorination, amination, and thiation. Examples of the non-natural base include 2′-fluoropyrimidine and 2′-O-methylpyrimidine, and specific examples thereof include 2′-fluorouracil, 2′-aminouracil, 2′-O-methyluracil, and 2-thiouracil. The nucleotide may be, for example, modified nucleotide, and examples of the modified nucleotide include 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′-thiated-uracil nucleotide residue, and 2′-thiated-cytosine nucleotide residue. The binding nucleic acid molecule (A) may include non-nucleotide such as PNA (peptide nucleic acid) or LNA (Locked Nucleic Acid), for example.
The catalyst nucleic acid molecule (D) is not limited as long as it exerts a catalytic function. The catalytic function is, for example, the catalytic function of an oxidation-reduction reaction. The oxidation-reduction reaction is not limited as long as it is a reaction in which electrons are transferred between two substrates in a course of generating a product from the substrates, for example. The kind of the oxidation-reduction reaction is not particularly limited. The catalytic function of the oxidation-reduction reaction can be, for example, activity which is the same as in enzyme and is, for example, specifically activity that is the same as in peroxidase (hereinafter referred to as “peroxidase-like activity”). The peroxidase activity can be, for example, horseradish peroxidase (HRP) activity. In the case where the catalyst nucleic acid molecule (D) is DNA such as described below, it can be called DNA enzyme or DNAzyme. In the case where the catalyst nucleic acid molecule (D) is RNA such as described below, it can be called RNA enzyme or RNAzyme.
The catalyst nucleic acid molecule (D) is preferably a nucleic acid that forms G-quartet (or G-tetrad), more preferably a nucleic acid that forms guanine quadruple (or G-quadruple). The G-tetrad is, for example, a plane structure of guanine tetramer, and the G-quadruplex is, for example, a structure in which plural G-tetrads are overlapped. The G-tetrad and the G-quadruplex are formed in a nucleic acid having a G-rich structural motif by repetition, for example. Examples of the G-tetrad include a parallel-type G-tetrad and an anti-parallel-type G-tetrad, and the G-tetrad is preferably a parallel-type G-tetrad. It is preferred that, in the nucleic acid element, stems are formed in the state where a target does not bind to the binding nucleic acid element (A), thereby inhibiting formation of G-tetrad in the catalyst nucleic acid molecule (D), and the formation of the stems is released by a binding of a target to the binding nucleic acid molecule (A), thereby forming G-tetrad in the catalyst nucleic acid molecule (D), for example.
The catalyst nucleic acid molecule (D) is preferably a nucleic acid capable of binding to porphyrin, specifically preferably a nucleic acid that forms G-tetrad and is capable of binding to porphyrin. It has been known that the nucleic acid having G-tetrad exerts the above-mentioned catalytic function of the oxidation-reduction reaction by forming a complex through binding of the nucleic acid with porphyrin, for example. It is preferred that, in the nucleic acid element, stems are formed in the state where a target does not bind to the binding nucleic acid molecule (A), thereby inhibiting a binding of the catalyst nucleic acid molecule (D) to porphyrin, and the formation of the stems are released by a binding of a target to the binding nucleic acid molecule (A), thereby binding the catalyst nucleic acid molecule (D) to porphyrin, for example. Specifically, it is preferred that, in the nucleic acid element, in the state where the binding nucleic acid molecule (A) does not bind to a target, formation of G-tetrad in the catalyst nucleic acid molecule (D) is inhibited, and a binding of the catalyst nucleic acid molecule (D) to porphyrin is inhibited, and by a binding of a target to the binding nucleic acid molecule (A), G-tetrad is formed in the catalyst nucleic acid molecule (D), and the catalyst nucleic acid molecule (D) binds to porphyrin, for example.
The porphyrin is not particularly limited, and examples thereof include unsubstituted porphyrin and a derivative thereof. Examples of the derivative include substituted porphyrin and a metal porphyrin obtained by forming a complex between porphyrin and a metal element. The substituted porphyrin can be, for example, N-methylmesoporphyrin. The metal porphyrin can be, for example, hemin that is a ferric complex. The porphyrin is, for example, preferably the metal porphyrin, more preferably hemin.
The catalyst nucleic acid molecule (D) is, for example, a single strand. The length of the catalyst nucleic acid molecule (D) is not particularly limited. The lower limit thereof is, for example, 11-mer, preferably 13-mer, more preferably 15-mer, and the upper limit thereof is, for example, 60-mer, preferably 36-mer, more preferably 18-mer.
Examples of the catalyst nucleic acid molecule (D) include DNAzymes disclosed in the following literatures (1) to (4) as DNAs having peroxidase activity.

    (1) Travascio et. al., Chem. Biol., 1998, vol. 5, p. 505-517,
    (2) Cheng et. al., Biochimistry, 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.


As a specific example of the catalyst nucleic acid molecule (D), there is a catalyst nucleic acid molecule including the following polynucleotide (d1), (d2), (d3), or (d4). The catalyst nucleic acid molecule (D) may be a molecule that is composed of or includes the polynucleotide. The catalyst nucleic acid molecule (D) including the polynucleotide (d1), (d2), (d3), or (d4) can also be referred to as a catalyst DNA molecule or DNAzyme, for example.
(d1) a polynucleotide composed of a base sequence of any of SEQ ID NOs: 11 to 31 and 66 to 85,
(d2) a polynucleotide that is composed of a base sequence obtained by substitution, deletion, addition, and/or insertion of at least one base in the base sequence of the polynucleotide (d1) and exerts a catalytic function of an oxidation-reduction reaction,

(d3) a polynucleotide that is composed of a base sequence with 50% or more identity with the base sequence of the polynucleotide (d1) and exerts a catalytic function of an oxidation-reduction reaction, and

(d4) a polynucleotide that is composed of a base sequence complementary to a base sequence that hybridizes to the base sequence of the polynucleotide (d1) under stringent conditions and exerts a catalytic function of an oxidation-reduction reaction.










EAD2



(SEQ ID NO: 11)



CTGGGAGGGAGGGAGGGA



 



c-Myc



(SEQ ID NO: 12)



TGAGGGTGGGGAGGGTGGGGAA



 



m_c-Myc-0527



(SEQ ID NO: 13)



TGAGGGGAGGGAGGGCGGGGAA



 



m_c-Myc-0579



(SEQ ID NO: 14)



TGAGGGGTGGGAGGGAGGGGAA



 



m_c-Myc-0580



(SEQ ID NO: 15)



TGAGGGGTGGGAGGGACGGGAA



 



m_c-Myc-0583



(SEQ ID NO: 16)



TGAGGGGTGGGAGGGTGGGGAA



 



m_c-Myc-0584



(SEQ ID NO: 17)



TGAGGGGTGGGAGGGTCGGGAA



 



neco0584



(SEQ ID NO: 18)



GGGTGGGAGGGTCGGG



 



m_c-Myc-0586



(SEQ ID NO: 19)



TGAGGGGTGGGAGGGGTGGGAA



 



m_c-Myc-0588



(SEQ ID NO: 20)



TGAGGGGTGGGAGGGGCGGGAA



 



m_c-Myc-0605



(SEQ ID NO: 21)



TGAGGGGTGGGTGGGCAGGGAA



 



m_c-Myc-0608



(SEQ ID NO: 22)



TGAGGGGTGGGTGGGCCGGGAA



 



m_c-Myc-0627



(SEQ ID NO: 23)



TGAGGGGTGGGCGGGAGGGGAA



 



m_c-Myc-0632



(SEQ ID NO: 24)



TGAGGGGTGGGCGGGTCGGGAA



 



m_c-Myc-0706



(SEQ ID NO: 25)



TGAGGGGCGGGAGGGATGGGAA



 



m_c-Myc-0711



(SEQ ID NO: 26)



TGAGGGGCGGGAGGGTGGGGAA



 



m_c-Myc-0712



(SEQ ID NO: 27)



TGAGGGGCGGGAGGGTCGGGAA



 



m_EAD2-0032



(SEQ ID NO: 28)



CTGGGTGGGCGGGCGGGA



 



m_c-Myc-0520



(SEQ ID NO: 29)



TGAGGGGAGGGAGGGTCGGGAA



 



m_c-Myc-0714



(SEQ ID NO: 30)



TGAGGGGCGGGAGGGGTGGGAA



 



m_TA-0420



(SEQ ID NO: 31)



GGGCGGGAGGGAGGG



 



SEQ ID NO: 66



GTGGGTCATTGTGGGTGGGTGTGG



 



SEQ ID NO: 67



GTGGGTAGGGCGGGTTGG



 



SEQ ID NO: 68



GGTTGGTGTGGTTGG



 



SEQ ID NO: 69



GGGGTTGGGGTGTGGGGTTGGGG



 



SEQ ID NO: 70



AGGGTTAGGGTTAGGGTTAGGG



 



SEQ ID NO: 71



GGGGTTTTGGGGTTTTGGGGTTTTGGGG



 



SEQ ID NO: 72



GGGCGCGGGAGGAAGGGGGCGGG



 



SEQ ID NO: 73



GTGGGTAGGGCGGTTGG



 



SEQ ID NO: 74



CGAGGTGGGTGGGTGGGA



 



SEQ ID NO: 75



CTGGGTGGGTGGGTGGGA



 



SEQ ID NO: 76



CTGGGAGGGAGGGAGGGA



 



SEQ ID NO: 77



CTGGGCGGGCGGGCGGGA



 



SEQ ID NO: 78



CTGGGTTGGGTTGGGTTGGGA



 



SEQ ID NO: 79



CTGGGGTGGGGTGGGGTGGGGA



 



SEQ ID NO: 80



GGGCGGGCCGGGGGCGGG



 



SEQ ID NO: 81



TGAGGGTGGGGAGGGTGGGGAA



 



SEQ ID NO: 82



CGGGCGGGCGCGAGGGAGGGG



 



SEQ ID NO: 83



GGGAGGGAGAGGGGGCGGG



 



SEQ ID NO: 84



GGGCGGGCGCGGGCGGG



 



SEQ ID NO: 85



GGGTAGGGCGGGTTGGG






In the polynucleotide (d2), “at least one” is not particularly limited as long as the polynucleotide (d2) exerts the catalytic function of the oxidation-reduction reaction. The number of substituted bases is, in the base sequence of the polynucleotide (d1), for example, from 1 to 5, preferably from 1 to 4, more preferably from 1 to 3, yet more preferably 1 or 2, particularly preferably 1. The number of added or inserted bases is, in the base sequence of the polynucleotide (d1), for example, from 1 to 5, preferably from 1 to 4, more preferably from 1 to 3, yet more preferably 1 or 2, particularly preferably 1. The number of deleted bases is, in the base sequence of the polynucleotide (d1), for example, from 1 to 5, preferably from 1 to 4, more preferably from 1 to 3, yet more preferably 2 or 1, particularly preferably 1.
In the polynucleotide (d3), the identity is, to the base sequence of the polynucleotide (d1), for example, 70% or more, preferably 80% or more, more preferably 90% or more, yet more preferably 95% or more, 96% or more, 97% or more, 98% or more, particularly preferably 99% or more. The identity can be calculated using BLAST or the like under default conditions, for example.
In the polynucleotide (d4), “hybridization under stringent conditions” is the same as mentioned above.
The catalyst nucleic acid molecule (D) is not limited by the examples of the polynucleotides (d1) to (d4) as long as it is a nucleic acid molecule that exerts the catalytic function as mentioned above.
The catalyst nucleic acid molecule (D) is, for example, a molecule including a nucleotide residue and may be a molecule composed of only or includes a nucleotide residue. The nucleotide is the same as mentioned above. The catalyst nucleic acid molecule (D) may include, for example, only one kind of ribonucleotide, deoxyribonucleotide, and derivatives thereof, two or more kinds of them, or all of them. Specifically, the nucleic acid molecule may be DNA including deoxyribonucleotide and/or a derivative thereof, RNA including ribonucleotide and/or a derivative thereof, or chimera (DNA/RNA) including the former and the latter, for example.
The nucleotide can be described with reference to examples shown for the binding nucleic acid molecule (A). The catalyst nucleic acid molecule (D) may include non-nucleotide such as PNA (peptide nucleic acid) or LNA (Locked Nucleic Acid), for example.
In the present invention, examples of the nucleic acid element include the above-mentioned nucleic acid elements (I), (II), (II′), and (III). Three forms of them are described below. Each form can be described with reference to the descriptions of the other forms unless otherwise shown.
As mentioned above, the nucleic acid element (I) is a double-stranded nucleic acid element composed of a first strand and a second strand. The second strand is also referred to as a block strand. The first strand (ss1) includes a binding nucleic acid molecule (A), a loop-forming sequence (L1), and a catalyst nucleic acid molecule (D) linked in this order. The second strand (ss2) includes a stem-forming sequence (SA), a loop-forming sequence (L2), and a stem-forming sequence (SD) linked in this order. A terminal region of the binding nucleic acid molecule (A) in the first strand (ss1) on the loop-forming sequence (L1) side is complementary to the stem-forming sequence (SA) in the second strand (ss2). A terminal region of the catalyst nucleic acid molecule (D) in the first strand (ss1) on the loop-forming sequence (L1) side is complementary to the stem-forming sequence (SD) in the second strand (ss2). The loop-forming sequence (L1) in the first strand (ss1) is non-complementary to the loop-forming sequence (L2) in the second strand (ss2). In the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited by stem formation in each of the stem-forming sequences (SA) and (SD). The inhibition of the catalytic function occurs by caging the catalyst nucleic acid molecule through this stem formation. That is, by the stem formation, the catalyst nucleic acid molecule (D) cannot have a structure with which the catalytic function is exerted, thereby inhibiting the catalytic function. On the other hand, in the presence of a target, the stem formation in each of the stem-forming sequences (SA) and (SD) is released by a binding of the target with the binding nucleic acid molecule (A), and the catalytic function of the catalyst nucleic acid molecule (D) is exerted. The exertion of the catalytic function occurs by releasing the stem formation and the caging of the catalyst nucleic acid molecule, for example. That is, by releasing the stem formation, the catalyst nucleic acid molecule (D) has an original structure with which the catalytic function is exerted, thereby exerting the catalytic function. Note here that the present invention is not limited by these mechanisms.
As described above, in the nucleic acid element (I), in the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited (switched OFF) by the stem formation, and in the presence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is exerted (switched ON) by releasing the stem formation. Specifically, in the nucleic acid element (I), for example, in the absence of a target, a terminal region of the binding nucleic acid molecule (A) in the first strand (ss1) on the loop-forming sequence (L1) side and the stem-forming sequence (SA) in the second strand (ss2) form a stem, a terminal region of the catalyst nucleic acid molecule (D) in the first strand (ss1) on the loop-forming sequence (L1) side and the stem-forming sequence (SD) in the second strand (ss2) form a stem, and the loop-forming sequences (L1) and (L2) form an internal loop between the two stems. In the present invention, “being complementary” may be, for example, the state where two regions aligned with each other are completely complementary to each other or the state where the two regions are complementary to each other to the extent that the two regions can form stems (hereinafter the same). Moreover, in the present invention, “being non-complementary” may be, for example, the state where two regions aligned with each other are completely non-complementary to each other or the state where the two regions are non-complementary to each other to the extent that the two regions can form an internal loop (hereinafter the same).
The schematic views of FIGS. 1A and 1B show the state of the nucleic acid element (I) in the absence of a target. The form shown in FIG. 1A and the form shown in FIG. 1B are reversed from each other in terms of the directions of the first strand (ss1) and the second strand (ss2). In FIGS. 1A and 1B, each arrow indicates the direction from the 5′ side to the 3′ side (hereinafter the same).
In each of FIGS. 1A and 1B, an upper strand is the first strand (ss1), A represents the binding nucleic acid molecule (A), L1 represents the loop-forming sequence (L1), and D represents the catalyst nucleic acid molecule (D). A lower strand is the second strand (ss2), SA represents the stem-forming sequence (SA), L2 represents the loop-forming sequence (L2), and SD represents the stem-forming sequence (SD). As shown in each of FIGS. 1A and 1B, in the absence of a target, two stems are formed at the respective two positions between the first strand (ss1) and the second strand (ss2), and an internal loop is formed between the stems. Specifically, a stem is formed between a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side, a stem is formed between a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L1) side and the stem-forming sequence (SD), and an internal loop is formed between the loop-forming sequences (L1) and (L2). In the presence of a target, the formation of the two stems is released by a binding of a target to the binding nucleic acid molecule (A), thereby exerting catalytic activity of the catalyst nucleic acid molecule (D).
The order of each component in the nucleic acid element (I) is not particularly limited. As shown in FIG. 1A, it is preferred that the first strand (ss1) includes, from the 5′ side thereof, the binding nucleic acid molecule (A), the loop-forming sequence (L1), and the catalyst nucleic acid molecule (D) linked in this order, and the second strand (ss2) includes, from the 3′ side thereof, the stem-forming sequence (SA), the loop-forming sequence (L2), and the stem-forming sequence (SD) linked in this order, for example. In this case, it is preferred that a 3′ terminal region of the binding nucleic acid molecule (A) in the first strand (ss1) is complementary to the stem-forming sequence (SA) in the second strand (ss2), and a 5′ terminal region of the catalyst nucleic acid molecule (D) in the first strand (ss1) is complementary to the stem-forming sequence (SD) in the second strand (ss2). As shown in FIG. 1B, for example, the first strand (ss1) may include, from the 3′ side thereof, the binding nucleic acid molecule (A), the loop-forming sequence (L1), and the catalyst nucleic acid molecule (D) linked in this order, and the second strand (ss2) may include, from the 5′ side thereof, the stem-forming sequence (SA), the loop-forming sequence (L2), and the stem-forming sequence (SD) linked in this order. In this case, it is preferred that a 5′ terminal region of the binding nucleic acid molecule (A) in the first strand (ss1) is complementary to the stem-forming sequence (SA) in the second strand (ss2), and a 3′ terminal region of the catalyst nucleic acid molecule (D) in the first strand (ss1) is complementary to the stem-forming sequence (SD) in the second strand (ss2). It is assumed that the nucleic acid element (I) can perform detection with superior sensitivity by forming an internal loop as described above. The present invention, however, is not limited by the assumption.
In the nucleic acid element (I), the length of the internal loop is not particularly limited. The length of each of the loop-forming sequence (L1) in the first strand (ss1) and the loop-forming sequence (L2) in the second strand (ss2) is, for example, from 0- to 30-mer, preferably from 1- to 30-mer, more preferably from 1- to 15-mer, yet more preferably from 1- to 6-mer. The lengths of the loop-forming sequences (L1) and (L2) may be identical to or different from each other, for example. In the latter case, the difference in length is not particularly limited and is, for example, from 1 to 10 bases, preferably 1 or 2 bases, more preferably 1 base. The nucleic acid element (I) may include only either one of the loop-forming sequences (L1) and (L2). It is assumed that the nucleic acid element (I) can perform detection with superior sensitivity by forming an internal loop as described above. The present invention, however, is not limited by the assumption.
In the nucleic acid element (I), the length of each stem is not particularly limited. The length of the stem can be adjusted by the lengths of the stem-forming sequences (SA) and (SD) in the second strand (ss2), for example. The length of the stem-forming sequence (SA) is, for example, from 0- to 60-mer, preferably from 0- to 10-mer, more preferably from 1- to 6-mer. The length of the stem-forming sequence (SD) is, for example, from 0- to 30-mer, from 1- to 30-mer, preferably from 0- to 10-mer, from 1- to 10-mer, more preferably from 1- to 6-mer.
In the nucleic acid element (I), 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, from 40- to 200-mer, preferably from 42- to 100-mer, more preferably from 45- to 60-mer. The length of the second strand (ss2) is, for example, from 4- to 120-mer, preferably from 5- to 25-mer, more preferably from 10- to 15-mer.
Specific examples of the nucleic acid element (I) using, as the binding nucleic acid molecule (A), an ATP/AMP aptamer that is the ATP/AMP binding nucleic acid (A) are shown below. The present invention, however, is not limited thereby. Examples of the first strand (ss1) are shown below. In each of the following sequences, the underlined portion on the 5′-side indicates an ATP/AMP aptamer (A in each FIGS. 1A and 1B) of SEQ ID NO: 1, the poly dT indicates the loop-forming sequence (L1 in each of FIGS. 1A and 1B), and the underlined portion on the 3′ side indicates DNAzyme (D in each of FIGS. 1A and 1B) of SEQ ID NO: 18 (neco0584).







AMP.neco.D0.A0


(SEQ ID NO: 2)


5′-CCTGGGGGAGTATTGCGGAGGAAGGTTTTTTTTGGGTGGGAGGGTCG


 


GG-3′






Examples of the second strand (ss2) to be paired with the first strand (ss1) are shown below. In each of the following sequences, the underlined portion on the 5′ side indicates the stem-forming sequence (SD in each of FIGS. 1A and 1B) complementary to a 5′ side region of the DNAzyme in the first strand (ss1), the poly dT indicates the loop-forming sequence (L2 in each of FIGS. 1A and 1B), and the underlined portion on the 3′ side indicates the stem-forming sequence (SA in each of FIGS. 1A and 1B) complementary to a 3′ side region of the ATP/AMP aptamer.









AMP.neco.D5.A5



(SEQ ID NO: 3)



5′-CACCCTTTTTTTTCCTTC-3′



 



AMP.neco.D5.A6



(SEQ ID NO: 4)



5′-CACCCTTTTTTTTCCTTCC-3′



 



AMP.neco.D5.A7



(SEQ ID NO: 5)



5′-CACCCTTTTTTTTCCTTCCT-3′



 



AMP.neco.D5.A8



(SEQ ID NO: 6)



5′-CACCCTTTTTTTTCCTTCCTC-3′



 



AMP.neco.D6.A5



(SEQ ID NO: 7)



5′-CCACCCTTTTTTTTCCTTC-3′



 



AMP.neco.D6.A6



(SEQ ID NO: 8)



5′-CCACCCTTTTTTTTCCTTCC-3′



 



AMP.neco.D6.A7



(SEQ ID NO: 9)



5′-CCACCCTTTTTTTTCCTTCCT-3′



 



AMP.neco.D6.A8



(SEQ ID NO: 10)



5′-CCACCCTTTTTTTTCCTTCCTC-3′



 



AMP.neco.D7.A5



(SEQ ID NO: 32)



5′-CCCACCCTTTTTTTTCCTTC-3′



 



AMP.neco.D7.A6



(SEQ ID NO: 33)



5′-CCCACCCTTTTTTTTCCTTCC-3′



 



AMP.neco.D7.A7



(SEQ ID NO: 34)



5′-CCCACCCTTTTTTTTCCTTCCT-3′



 



AMP.neco.D7.A8



(SEQ ID NO: 35)



5′-CCCACCCTTTTTTTTCCTTCCTC-3′



 



AMP.neco.D8.A5



(SEQ ID NO: 36)



5′-TCCCACCCTTTTTTTTCCTTC-3′



 



AMP.neco.D8.A6



(SEQ ID NO: 37)



5′-TCCCACCCTTTTTTTTCCTTCC-3′



 



AMP.neco.D8.A7



(SEQ ID NO: 38)



5′-TCCCACCCTTTTTTTTCCTTCCT-3′



 



AMP.neco.D8.A8



(SEQ ID NO: 39)



5′-TCCCACCCTTTTTTTTCCTTCCTC-3′






The nucleic acid element (II) is, as mentioned above, a single-stranded nucleic acid element and includes a binding nucleic acid molecule (A), a loop-forming sequence (L1), a stem-forming sequence (SD), a catalyst nucleic acid molecule (D), a loop-forming sequence (L2), and a stem-forming sequence (SA) linked in this order. A terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side is complementary to the stem-forming sequence (SA), a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side is complementary to the stem-forming sequence (SD), and the loop-forming sequence (L1) is non-complementary to the loop-forming sequence (L2).
In the nucleic acid element (II), in the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited by stem formation in each of the stem-forming sequences (SA) and (SD). The inhibition of the catalytic function occurs by caging the catalyst nucleic acid molecule through such stem formation caused by self-association. That is, by the stem formation, the catalyst nucleic acid molecule (D) cannot have a structure with which the catalytic function is exerted, thereby inhibiting the catalytic function. On the other hand, in the presence of a target, the stem formation in each of the stem-forming sequences (SA) and (SD) is released by a binding of the target with the binding nucleic acid molecule (A), and the catalytic function of the catalyst nucleic acid molecule (D) is exerted. The exertion of the catalytic function occurs by releasing the stem formation caused by the self-association and the caging of the catalyst nucleic acid molecule, for example. That is, by releasing the stem formation, the catalyst nucleic acid molecule (D) has an original structure with which the catalytic function is exerted, thereby exerting the catalytic function. Note here that the present invention is not limited by these mechanisms.
As described above, as in the nucleic acid element (I), in the nucleic acid element (II), in the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited (switched OFF) by the stem formation, and in the presence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is exerted (switched ON) by releasing the stem formation. Specifically, in the nucleic acid element (II), for example, in the absence of a target, a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side and the stem-forming sequence (SA) form a stem, a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side and the stem-forming sequence (SD) form a stem, and the loop-forming sequences (L1) and (L2) form an internal loop between the two stems.
The schematic views of FIGS. 2A and 2B show the state of the nucleic acid element (II) in the absence of a target. The form shown in FIG. 2A and the form shown in FIG. 2B are reversed from each other in terms of the direction.
In each of FIGS. 2A and 2B, A represents the binding nucleic acid molecule (A), L1 represents the loop-forming sequence (L1), SD represents the stem-forming sequence (SD), D represents the catalyst nucleic acid molecule (D), L2 represents the loop-forming sequence (L2), and SA represents the stem-forming sequence (SA). As shown in each of FIGS. 2A and 2B, in the absence of a target, two stems are formed at the respective two positions by self-annealing of the nucleic acid element (II), and an internal loop is formed between the stems. Specifically, a stem is formed between a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side, a stem is formed between a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L1) side and the stem-forming sequence (SD), and an internal loop is formed between the loop-forming sequences (L1) and (L2). In the presence of a target, the formation of the two stems is released by a binding of a target to the binding nucleic acid molecule (A), thereby exerting catalytic activity of the catalyst nucleic acid molecule (D).
The order of each component in the nucleic acid element (II) is not particularly limited. As shown in FIG. 2B, it is preferred that the nucleic acid element (II) includes, from the 3′ side thereof, the binding nucleic acid molecule (A), the loop-forming sequence (L1), the stem-forming sequence (SD), the catalyst nucleic acid molecule (D), the loop-forming sequence (L2), and the stem-forming sequence (SA) linked in this order, for example. In this case, it is preferred that a 5′ terminal region of the binding nucleic acid molecule (A) is complementary to the stem-forming sequence (SA), and a 5′ terminal region of the catalyst nucleic acid molecule (D) is complementary to the stem-forming sequence (SD). As shown in FIG. 2A, the nucleic acid element (II) may include, from the 5′ side thereof, the binding nucleic acid molecule (A), the loop-forming sequence (L1), the stem-forming sequence (SD), the catalyst nucleic acid molecule (D), the loop-forming sequence (L2), and the stem-forming sequence (SA) linked in this order, for example. In this case, it is preferred that a 3′ terminal region of the binding nucleic acid molecule (A) is complementary to the stem-forming sequence (SA), and a 3′ terminal region of the catalyst nucleic acid molecule (D) is complementary to the stem-forming sequence (SD).
In the nucleic acid element (II), the length of the internal loop is not particularly limited. The length of each of the loop-forming sequences (L1) and (L2) is, for example, from 0- to 30-mer, preferably from 1- to 30-mer, more preferably from 1- to 15-mer, yet more preferably from 1- to 6-mer. The lengths of the loop-forming sequences (L1) and (L2) may be identical to or different from each other, for example. In the latter case, the difference in length is not particularly limited and is, for example, from 1 to 10 bases, preferably 1 or 2 bases, more preferably 1 base. The nucleic acid element (II) may include only either one of the loop-forming sequences (L1) and (L2). It is assumed that the nucleic acid element (II) can perform detection with superior sensitivity by forming an internal loop as described above. The present invention, however, is not limited by the assumption.
In the nucleic acid element (II), the length of each stem is not particularly limited. The length of the stem can be adjusted by the lengths of the stem-forming sequences (SA) and (SD), for example. The length of the stem-forming sequence (SA) is, for example, from 0- to 60-mer, from 1- to 60-mer, preferably from 1- to 10-mer, more preferably from 1- to 7-mer. The length of the stem-forming sequence (SD) is, for example, from 0- to 30-mer, from 1- to 30-mer, preferably from 0- to 10-mer, from 1- to 10-mer, more preferably from 0- to 7-mer, from 1- to 7-mer.
The length of the nucleic acid element (II) is not particularly limited and is, for example, from 40- to 120-mer, preferably from 45- to 100-mer, more preferably from 50- to 80-mer.
Specific examples of the nucleic acid element (II) using, as the binding nucleic acid molecule (A), an ATP/AMP aptamer that is the ATP/AMP binding nucleic acid (A) are shown below. The present invention, however, is not limited thereby. In each of the following sequences, from the 5′ side thereof toward the 3′ side thereof, the sequence indicated by lower-case letters indicates the stem-forming sequence (SA), the poly dT indicated by capital letters indicates the loop-forming sequence (L2), the underlined portion on the 5′ side indicates DNAzyme (D) of SEQ ID NO: 11 (EAD2), the sequence indicated by lower-case letters indicates the stem-forming sequence (SD), the poly dT indicated by capital letters indicates the loop-forming sequence (L1), and the underlined portion on the 3′ side indicates an ATP/AMP aptamer (A) of SEQ ID NO: 1.







AMP.D3.A2


(SEQ ID NO: 40)


5′-ggTTTCTGGGAGGGAGGGAGGGAcagTTTCCTGGGGGAGTATTGCGG


 


AGGAAGG-3′


 


AMP.D3.A3


(SEQ ID NO: 41)


5′-aggTTTCTGGGAGGGAGGGAGGGAcagTTTCCTGGGGGAGTATTGCG


 


GAGGAAGG-3′


 


AMP.D3.A4


(SEQ ID NO: 42)


5′-caggTTTCTGGGAGGGAGGGAGGGAcagTTTCCTGGGGGAGTATTGC


 


GGAGGAAGG-3′


 


AMP.D3.A5


(SEQ ID NO: 43)


5′-ccaggTTTCTGGGAGGGAGGGAGGGAcagTTTCCTGGGGGAGTATTG


 


CGGAGGAAGG-3′


 


AMP.D4.A2


(SEQ ID NO: 44)


5′-ggTTTCTGGGAGGGAGGGAGGGAccagTTTCCTGGGGGAGTATTGCG


 


GAGGAAGG-3′


 


AMP.D4.A3


(SEQ ID NO: 45)


5′-aggTTTCTGGGAGGGAGGGAGGGAccagTTTCCTGGGGGAGTATTGC


 


GGAGGAAGG-3′


 


AMP.D4.A4


(SEQ ID NO: 46)


5′-caggTTTCTGGGAGGGAGGGAGGGAccagTTTCCTGGGGGAGTATTG


 


CGGAGGAAGG-3′


 


AMP.D4.A5


(SEQ ID NO: 47)


5′-ccaggTTTCTGGGAGGGAGGGAGGGAccagTTTCCTGGGGGAGTATT


 


GCGGAGGAAGG-3′


 


AMP.D5.A2


(SEQ ID NO: 48)


5′-ggTTTCTGGGAGGGAGGGAGGGAcccagTTTCCTGGGGGAGTATTGC


 


GGAGGAAGG-3′


 


AMP.D5.A3


(SEQ ID NO: 49)


5′-aggTTTCTGGGAGGGAGGGAGGGAcccagTTTCCTGGGGGAGTATTG


 


CGGAGGAAGG-3′


 


AMP.D5.A4


(SEQ ID NO: 50)


5′-caggTTTCTGGGAGGGAGGGAGGGAcccagTTTCCTGGGGGAGTATT


 


GCGGAGGAAGG-3′


 


AMP.D5.A5


(SEQ ID NO: 51)


5′-ccaggTTTCTGGGAGGGAGGGAGGGAcccagTTTCTGGGGGGAGTAT


 


TGCGGAGGAAGG-3′






In each of the following sequences, from the 5′ side thereof toward the 3′ side thereof, the sequence indicated by lower-case letters indicates the stem-forming sequence (SA), the poly dT indicated by capital letters indicates the loop-forming sequence (L2), the underlined portion on the 5′ side indicates DNAzyme (D) of SEQ ID NO: 18 (neco0584), the sequence indicated by lower-case letters indicates the stem-forming sequence (SD), the poly dT indicated by capital letters indicates the loop-forming sequence (L1), and the underlined portion on the 3′ side indicates an ATP/AMP aptamer (A) of SEQ ID NO: 1.







AMP.neco.D3.A2


(SEQ ID NO: 52)


5′-ggTTTGGGTGGGAGGGTCGGGcccTTTCCTGGGGGAGTATTGCGGAG


 


GAAGG-3′


 


AMP.neco.D3.A3


(SEQ ID NO: 53)


5′-aggTTTGGGTGGGAGGGTCGGGcccTTTCCTGGGGGAGTATTGCGGA


 


GGAAGG-3′


 


AMP.neco.D3.A4


(SEQ ID NO: 54)


5′-caggTTTGGGTGGGAGGGTCGGGcccTTTCCTGGGGGAGTATTGCGG


 


AGGAAGG-3′


 


AMP.neco.D3.A5


(SEQ ID NO: 55)


5′-ccaggTTTGGGTGGGAGGGTCGGGcccTTTCCTGGGGGAGTATTGCG


 


GAGGAAGG-3′


 


AMP.neco.D4.A2


(SEQ ID NO: 56)


5′-ggTTTGGGTGGGAGGGTCGGGacccTTTCCTGGGGGAGTATTGCGGA


 


GGAAGG-3′


 


AMP.neco.D4.A3


(SEQ ID NO: 57)


5′-aggTTTGGGTGGGAGGGTCGGGacccTTTCCTGGGGGAGTATTGCGG


 


AGGAAGG-3′


 


AMP.neco.D4.A4


(SEQ ID NO: 58)


5′-caggTTTGGGTGGGAGGGTCGGGacccTTTCCTGGGGGAGTATTGCG


 


GAGGAAGG-3′


 


AMP.neco.D4.A5


(SEQ ID NO: 59)


5′-ccaggTTTGGGTGGGAGGGTCGGGacccTTTCCTGGGGGAGTATTGC


 


GGAGGAAGG-3′


 


AMP.neco.D5.A2


(SEQ ID NO: 60)


5′-ggTTTGGGTGGGAGGGTCGGGcacccTTTCCTGGGGGAGTATTGCGG


 


AGGAAGG-3′


 


AMP.neco.D5.A3


(SEQ ID NO: 61)


5′-aggTTTGGGTGGGAGGGTCGGGcacccTTTCCTGGGGGAGTATTGCG


 


GAGGAAGG-3′


 


AMP.neco.D5.A4


(SEQ ID NO: 62)


5′-caggTTTGGGTGGGAGGGTCGGGcacccTTTCCTGGGGGAGTATTGC


 


GGAGGAAGG-3′


 


AMP.neco.D5.A5


(SEQ ID NO: 63)


5′-ccaggTTTGGGTGGGAGGGTCGGGcacccTTTCCTGGGGGAGTATTG


 


CGGAGGAAGG-3′


 


AMP.neco.D6.A2


(SEQ ID NO: 64)


5′-ggTTTGGGTGGGAGGGTCGGGccacccTTTCCTGGGGGAGTATTGCG


 


GAGGAAGG-3′






The nucleic acid element (II′) is a single-stranded nucleic acid element having a positional relationship in which the binding nucleic acid molecule (A) is interchanged with the catalyst nucleic acid molecule (D), the stem-forming sequence (SA) is interchanged with the stem-forming sequence (SD), and the loop-forming sequence (L1) is interchanged with the loop-forming sequence (L2) in the nucleic acid element (II). The nucleic acid element (II′) can be described with reference to the description of the nucleic acid element (II) unless otherwise particularly described.
As mentioned above, the nucleic acid element (II′) includes a catalyst nucleic acid molecule (D), a loop-forming sequence (L2), a stem-forming sequence (SA), a binding nucleic acid molecule (A), a loop-forming sequence (L1), and a stem-forming sequence (SD) linked in this order. A terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side is complementary to the stem-forming sequence (SD), and a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side is complementary to the stem-forming sequence (SA).
In the nucleic acid element (II′), in the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited by stem formation in each of the stem-forming sequences (SA) and (SD). The inhibition of the catalytic function occurs by caging the catalyst nucleic acid molecule through such stem formation caused by self-association. That is, by the stem formation, the catalyst nucleic acid molecule (D) cannot have an original structure with which the catalytic function is exerted, thereby inhibiting the catalytic function. On the other hand, in the presence of a target, the stem formation in each of the stem-forming sequences (SA) and (SD) is released by a binding of the target with the binding nucleic acid molecule (A), and the catalytic function of the catalyst nucleic acid molecule (D) is exerted. The exertion of the catalytic function occurs by releasing the stem formation caused by the self-association and the caging of the catalyst nucleic acid molecule, for example. That is, by releasing the stem formation, the catalyst nucleic acid molecule (D) has an original structure with which the catalytic function is exerted, thereby exerting the catalytic function. Note here that the present invention is not limited by these mechanisms.
As in the nucleic acid element (II), in the nucleic acid element (II′), in the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited (switched OFF) by the stem formation, and in the presence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is exerted (switched ON) by releasing the stem formation. Specifically, in the nucleic acid element (II′), for example, in the absence of a target, a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side and the stem-forming sequence (SA) form a stem, a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side and the stem-forming sequence (SD) form a stem, and the loop-forming sequences (L1) and (L2) form an internal loop between the two stems.
The schematic views of FIGS. 3A and 3B show the state of the nucleic acid element (II′) in the absence of a target. The form shown in FIG. 3A and the form shown in FIG. 3B are reversed from each other in terms of the direction.
In each of FIGS. 3A and 3B, A represents the binding nucleic acid molecule (A), L1 represents the loop-forming sequence (L1), D represents the catalyst nucleic acid molecule (D), L2 represents the loop-forming sequence (L2), SA represents the stem-forming sequence (SA), and SD represents the stem-forming sequence (SD). As shown in each of FIGS. 3A and 3B, in the absence of a target, two stems are formed at the respective two positions by self-annealing of the nucleic acid element (II′), and an internal loop is formed between the stems. Specifically, a stem is formed between a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side, a stem is formed between a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side and the stem-forming sequence (SD), and an internal loop is formed between the loop-forming sequences (L1) and (L2). In the presence of a target, the formation of the two stems is released by a binding of a target to the binding nucleic acid molecule (A), thereby exerting catalytic activity of the catalyst nucleic acid molecule (D).
The order of each component in the nucleic acid element (II′) is not particularly limited. As shown in FIG. 3B, it is preferred that the nucleic acid element (II′) includes, from the 3′ side thereof, the catalyst nucleic acid molecule (D), the loop-forming sequence (L2), the stem-forming sequence (SA), the binding nucleic acid molecule (A), the loop-forming sequence (L1), and the stem-forming sequence (SD) linked in this order, for example. In this case, it is preferred that a 5′ terminal region of the binding nucleic acid molecule (A) is complementary to the stem-forming sequence (SA), and a 5′ terminal region of the catalyst nucleic acid molecule (D) is complementary to the stem-forming sequence (SD). As shown in FIG. 3A, the nucleic acid element (II′) may include, from the 5′ side thereof, the catalyst nucleic acid molecule (D), the loop-forming sequence (L2), the stem-forming sequence (SA), the binding nucleic acid molecule (A), the loop-forming sequence (L1), and the stem-forming sequence (SD) linked in this order, for example. In this case, it is preferred that a 3′ terminal region of the binding nucleic acid molecule (A) is complementary to the stem-forming sequence (SA), and a 3′ terminal region of the catalyst nucleic acid molecule (D) is complementary to the stem-forming sequence (SD).
The nucleic acid element (III) is, as mentioned above, a single-stranded nucleic acid element and includes a catalyst nucleic acid molecule (D), an intervening sequence (I), and a binding nucleic acid molecule (A) linked in this order. The intervening sequence (I) is non-complementary to the catalyst nucleic acid molecule (D) and the binding nucleic acid molecule (A). In the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited. On the other hand, in the presence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is exerted.
As in the nucleic acid elements (I), (II), and (II′), in the nucleic acid element (III), in the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited (switched OFF), and in the presence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is exerted (switched ON).
The schematic views of FIGS. 4A and 4B show the state of the nucleic acid element (III) in the absence of a target. In each of FIGS. 4A and 4B, A represents the binding nucleic acid molecule (A), I represents the intervening sequence (I), and D represents the catalyst nucleic acid molecule (D). The form shown in FIG. 4A and the form shown in FIG. 4B are reversed from each other in terms of the direction.
The reason why, in the nucleic acid element (III), the catalytic function of the catalyst nucleic acid molecule (D) is inhibited in the absence of a target and is exerted in the presence of a target can be assumed as follows, for example. The present invention, however, is not limited by the assumption. In the nucleic acid element (III), in the absence of a target, a part of the catalyst nucleic acid molecule (D) and a part of the binding nucleic acid molecule (A) interact with each other, and the catalyst nucleic acid molecule (D) forms non-G quartet. In the presence of a target, the main form of the nucleic acid element (III) becomes close to the original structure of the binding nucleic acid element (A), i.e., a three-dimensional structure with which the binding nucleic acid element (A) binds to a target, and the entire structure changes. Thus, the catalyst nucleic acid molecule (D) forms G-quartet, and the catalytic function is exerted.
The order of each component in the nucleic acid element (III) is not particularly limited. As shown in FIG. 4A, it is preferred that the nucleic acid element (III) includes, from the 5′ side thereof, the catalyst nucleic acid molecule (D), the intervening sequence (I), and the binding nucleic acid molecule (A) linked in this order, for example. As shown in FIG. 4B, the nucleic acid element (III) includes, from the 3′ side thereof, the catalyst nucleic acid molecule (D), the intervening sequence (I), and the binding nucleic acid molecule (A) linked in this order, for example.
In the nucleic acid element (III), the length of the intervening sequence (I) is not particularly limited. The length of the intervening sequence (I) is, for example, for example, from 0- to 30-mer, from 1- to 30-mer, preferably from 0- to 25-mer, from 1- to 25-mer, more preferably from 0- to 20-mer, from 1- to 20-mer, yet more preferably from 0- to 10-mer, from 1- to 10-mer, particularly preferably from 0- to 8-mer, from 1- to 8-mer.
The length of the nucleic acid element (III) is not particularly limited. The length of the nucleic acid element (III) is, for example, from 30- to 120-mer, preferably from 35- to 80-mer, more preferably from 40- to 60-mer.
A specific example of the nucleic acid element (III) using, as a binding nucleic acid molecule (A), an ATP/AMP aptamer that is an ATP/AMP binding nucleic acid (A) is shown below. The present invention, however, is not limited thereby. In the following AMP.neco.D0.A0, the underlined portion on the 5′ side indicates DNAzyme (D in each of FIGS. 4A and 4B) of SEQ ID NO: 18 (neco0584), the underlined portion on the 3′ side indicates an ATP/AMP aptamer (A in each of FIGS. 4A and 4B) of SEQ ID NO: 1, and the poly dT positioned between the DNAzyme and the ATP/AMP aptamer indicates an intervening sequence (I in each of FIGS. 4A and 4B).







AMP.neco.D0.A0


(SEQ ID NO: 65)


5′-GGGTGGGAGGGTCGGGTTTTTTCCTGGGGGAGTATTGCGGAGGAAG


 


G-3′






In each of the nucleic acid elements (I), (II), (II′), and (III), the regions may be directly or indirectly linked, for example. In the case of the direct linking, for example, the regions are linked by a phosphodiester bond. In the case of the indirect linking, for example, the regions are linked via an intervening linker. The intervening linker can be, for example, a nucleic acid molecule composed of the above-mentioned nucleotide and/or non-nucleotide. The intervening linker is, for example, preferably a single strand.
The nucleic acid sensor of the present invention may be, for example, a sensor composed of only the nucleic acid element or a sensor further including any other component(s). The nucleic acid sensor of the present invention can also be referred to as a device for detecting SA, for example. One of the other component(s) can be, for example, a base material on/in which the nucleic acid element is arranged. The base material can be, for example, a base plate, a bead, a container such as a tube, or the like. In addition, one of the other component(s) can be, for example, a linker. The linker can be used to link between the nucleic acid element and the base material when the nucleic acid element is immobilized on the base material, for example. The arrangement of the nucleic acid sensor on the base material can be performed with reference to the description of the analysis device of the present invention described below.
In the nucleic acid element, the linking site with the linker is not particularly limited and is, for example, preferably either one of the terminals of the nucleic acid element. In the double-stranded nucleic acid element (I), the linking site is, for example, preferably either one of the terminals of the first strand (ss1) including the binding nucleic acid molecule (A) and the catalyst nucleic acid molecule (D) and/or either one of the terminals of the second strand (ss2). In each of the single-stranded nucleic acid elements (II), (II′), and (III), the linking site is, for example, preferably either one of the terminals. In this case, the linker is also referred to as a terminal linker. The linker can be, for example, a nucleic acid molecule composed of the above-mentioned nucleotide and/or non-nucleotide. The terminal linker is, for example, preferably a single strand.
The nucleic acid sensor of the present invention may be used in the state where the nucleic acid element is freed or in the state where the nucleic acid element is immobilized, for example. In the latter case, for example, the nucleic acid element is immobilized on the base material, which can be used as a device.
A method for using the nucleic acid sensor of the present invention is not particularly limited and can be used in the method for analyzing a target of the present invention as described below.
The analysis method of the present invention is, as mentioned above, a method for analyzing a target, including: a contact step of causing a sample containing a target to be in contact with the nucleic acid sensor according to the present invention; and a detection step of detecting the catalytic function of the catalyst nucleic acid molecule (D) in the nucleic acid sensor to detect a target in the sample.
The sample is not particularly limited. The sample may be, for example, either one of a sample containing a target and a sample in which whether or not it contains a target is unknown. The sample is, for example, preferably a liquid sample.
When the nucleic acid element in the state of being freed is used as the nucleic acid sensor of the present invention, it is preferred that the nucleic acid element and the sample are caused to be in contact with each other in a container such as a tube, for example. When the nucleic acid element in the state of being arranged on the base material is used as the nucleic acid sensor of the present invention, the sample can be caused to be in contact with the nucleic acid element on the base material, for example.
It is preferred that a signal generated by the catalytic function of the catalyst nucleic acid molecule (D) is detected in the detection step, for example. Examples of the signal include an optical signal and an electrochemical signal. Examples of the optical signal include a chromogenic signal, a luminescent signal, and a fluorescent signal.
It is preferred that the signal is generated from a substrate by the catalytic function of the catalyst nucleic acid molecule (D), for example. Thus, it is preferred that the detection step can be performed in the presence of a substrate appropriate to the catalytic function of the catalyst nucleic acid molecule (D), for example.
The substrate can be, for example, a substrate that generates a chromogenic, luminescent, or fluorescent product by the catalytic function or is a chromogenic, luminescent, or fluorescent substrate and generates a product that quenches its developed color, luminescence, or fluorescence by the catalytic function or generates a different chromogenic, luminescent, or fluorescent product by the catalytic function. According to such substrate, for example, the catalytic function can be detected by observing, as a signal, the presence or absence of developed color, luminescence, or fluorescence or the change in or the intensity of developed color, luminescence, or fluorescence by visual check. Alternatively, for example, the catalytic function can be detected by measuring, as a signal, absorbance, reflectance, or fluorescence intensity using an optical method. The catalytic function can be, for example, the above-mentioned catalytic function of the oxidation-reduction reaction.
In the case where the catalyst nucleic acid molecule (D) has the catalytic function of the oxidation-reduction reaction, the substrate can be, for example, a substrate that can perform electron transfer. In this case, for example, a product is generated from the substrate by the catalyst nucleic acid molecule (D), and in the course of the generation, electrons are transferred. This electron transfer can be electrochemically detected as an electrical signal by applying a voltage to an electrode, for example. The electrical signal can be detected by measuring the intensity of the electrical signal such as a current, for example.
The substrate is not particularly limited, and examples thereof include hydrogen peroxide, 3,3′,5,5′-Tetramethylbenzidine (TMB), 1,2-Phenylenediamine (OPD), 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic Acid Ammonium Salt (ABTS), 3,3′-Diaminobenzidine (DAB), 3,3′-Diaminobenzidine Tetrahydrochloride Hydrate (DAB4HCl), 3-Amino-9-ethylcarbazole (AEC), 4-Chloro-1-naphthol (4ClN), 2,4,6-Tribromo-3-hydroxybenzoic Acid, 2,4-Dichlorophenol, 4-Aminoantipyrine, 4-Aminoantipyrine Hydrochloride, and luminol.
In the detection step, the substrate may be, for example, supplied to the nucleic acid sensor before, at the same time as, or after causing the sample to be in contact with the nucleic acid sensor. It is preferred that the substrate is supplied to the nucleic acid sensor as a substrate liquid obtained by mixing the substrate in a liquid, for example. The liquid in which the substrate is mixed is, for example, preferably a buffer solution such as Tris-HCl. The concentration of the substrate in the substrate liquid is not particularly limited and is, for example, from 0.1 to 5 mmol/L, preferably from 0.5 to 2 mmol/L. The pH of the substrate liquid is, for example, from 6 to 9, preferably from 6.8 to 9.
In the detection step, the conditions of the reaction by the catalyst nucleic acid molecule (D) is not particularly limited. The temperature is, for example, from 15° C. to 37° C., and the time is, for example, from 10 to 900 seconds.
In the detection step, porphyrin may be present together in addition to the substrate, for example. There is known DNAzyme that exerts high oxidation-reduction activity by forming a complex with porphyrin, for example. Thus, in the present invention, for example, a complex between the catalyst nucleic acid molecule (D) and porphyrin may be formed by causing porphyrin to be present together to detect oxidation-reduction activity. The supply of porphyrin is not particularly limited and can be performed in the same manner as for the substrate.
The porphyrin is not particularly limited, and examples thereof include unsubstituted porphyrin and a derivative thereof. Examples of the derivative include substituted porphyrin and a metal porphyrin obtained by forming a complex between porphyrin and a metal element. The substituted porphyrin can be, for example, N-methylmesoporphyrin. The metal porphyrin can be, for example, hemin that is a ferric complex. The porphyrin is, for example, preferably the metal porphyrin, more preferably hemin.
According to the nucleic acid sensor of the present invention, a target can be detected as mentioned above. Specifically, for example, according to the nucleic acid sensor in which a target is ATP or AMP, and an ATP/AMP binding nucleic acid molecule (A) is used as the binding nucleic acid molecule (A), for example, contaminations such as microorganisms and food residues remaining in an object can be detected indirectly by detecting ATP or AMP as mentioned above, for example.
(Analysis Device and Analysis Method Using the Same)
The analysis device of the present invention is a device for analyzing a target, including: a base material; a nucleic acid sensor; and a detection part, wherein the nucleic acid sensor and the detection part are arranged in the base material, the nucleic acid sensor is the nucleic acid sensor according to the present invention, and the detection part is a detection part that detects the catalytic function of the catalyst nucleic acid molecule (D) in the nucleic acid sensor.
The analysis device of the present invention is characterized in that the nucleic acid sensor of the present invention is used, and the other configurations are not at all limited. The analysis device of the present invention can be described with reference to, for example, the description of the nucleic acid sensor of the present invention unless otherwise specifically described.
A method for arranging the nucleic acid sensor in the analysis device of the present invention is not particularly limited, and the nucleic acid element in the nucleic acid sensor may or may not be immobilized on the base material, for example. In the former case, the nucleic acid element may be directly or indirectly immobilized on the base material, for example. As the immobilization, a linkage by a chemical bond can be shown as an example. The indirect immobilization can have a form of immobilizing the nucleic acid element on the base material via a linker, for example. The linker can be, for example, the above-mentioned terminal linker. The arrangement of the nucleic acid element can be described with reference to the description of the nucleic acid sensor of the present invention, for example.
As the immobilization of the nucleic acid element, a known nucleic acid immobilization method can be employed other than the above-mentioned method, for example. The known nucleic acid immobilization method can be, for example, a method utilizing photolithography, and as a specific example, U.S. Pat. No. 5,424,186 can be used as a reference. The immobilization method can be, for example, a method in which the nucleic acid element is synthesized on the base material. This method can be, for example, a so-called spot method, and as a specific example, U.S. Pat. No. 5,807,522 or JP H10-503841 A can be used as a reference.
A site of the base material on which the nucleic acid element is arranged is not particularly limited and can have, for example, a form of arranging the nucleic acid element in the detection part.
The analysis device of the present invention may further include a reagent part, for example. The reagent part may be arranged in the detection part, for example. A reagent may be arranged in the reagent part in advance, or a reagent may be supplied in the reagent part when used. Examples of the reagent include the above-mentioned substrate and porphyrin.
In the analysis device of the present invention, the detection part is, as mentioned above, a detection part that detects the catalytic function of the catalyst nucleic acid molecule (D). The detection part is preferably a detection part that detects, as the catalytic function of the catalyst nucleic acid molecule (D), a signal generated by the catalytic function of the catalyst nucleic acid molecule (D). The signal can be, for example, as mentioned above, a signal generated from the substrate by the catalytic function of the catalyst nucleic acid molecule (D). The signal can be, for example, the above-mentioned optical signal or electrochemical signal.
In the case where the signal is an optical signal, the detection part is, for example, a detection part that detects an optical signal, and the detection part that detects an optical signal can be, for example, a detection part that detects absorbance, reflectance, fluorescence, or the like.
In the case where the signal is the electrochemical signal, the detection part includes an electrode system, for example. In this case, the detection part can be formed by arranging the electrode system on the surface of the base material, for example. A method for arranging the electrode is not particularly limited, and a known method can be employed, for example. Specific examples thereof include methods for forming a thin film such as a vapor deposition method, a sputtering method, a screen printing method, and a plating method. The electrode may be arranged directly or indirectly on the base material. The indirect arrangement can be, for example, an arrangement via another member.
The electrode system may be an electrode system including a working electrode and a counter electrode or an electrode system including a working electrode, a counter electrode, and a reference electrode, for example. The material of each electrode is not particularly limited, and examples thereof include platinum, silver, gold, and carbon. Examples of the working electrode and the counter electrode include a platinum electrode, a silver electrode, a gold electrode, and a carbon electrode. The reference electrode can be, for example, a silver/silver chloride electrode. The silver-silver chloride electrode can be formed by laminating a silver chloride electrode on a silver electrode, for example.
In the case where the analysis device of the present invention has the electrode system, the nucleic acid element is preferably arranged in the electrode system, more preferably arranged in the working electrode among the electrodes. In the case where the analysis device of the present invention includes the electrode system and the reagent part, the reagent part is preferably arranged on the electrode system, for example.
The analysis device of the present invention may include plural detection parts, for example. In this case, for example, in the analysis device, it is preferred that the surface of the base material is fractionated into matrix, and each fraction region is provided with each of the above-mentioned detection parts. In the analysis device of the present invention, the number of nucleic acid sensors arranged in one detection part is not particularly limited.
The base material is not particularly limited. The base material is, for example, preferably a base material having an insulating surface. The base material may be a base plate composed of an insulating material or a base material having, on the surface thereof, an insulating layer composed of an insulating material. The insulating material is not particularly limited, and examples thereof include known materials such as glass, ceramics, an insulating plastic, and paper. The insulating plastic is not particularly limited, and examples thereof include a silicone resin, a polyimide resin, an epoxy resin, and a fluorine resin.
The analysis method of the present invention is, as mentioned above, a method for analyzing a target, including: a contact step of causing a sample to be in contact with the analysis device according to the present invention; and a detection step of detecting the catalytic function of the catalyst nucleic acid molecule (D) in the detection part of the analysis device to detect a target in the sample.
The analysis method of the present intention is characterized in that the analysis device including the nucleic acid sensor of the present invention is used, and the other conditions are not at all limited. The analysis method of the present invention can be described with reference to the description of the analysis method in the description of the nucleic acid sensor of the present invention, for example.
(Analysis Reagent)
The analysis reagent of the present invention contains the nucleic acid sensor of the present invention. The analysis reagent of the present invention is characterized in that it contains the nucleic acid sensor, and the other configurations are not at all limited.
The analysis reagent of the present invention may further contain, in addition to the nucleic acid sensor, a component(s) such as the substrate, the porphyrin, the buffer solution, and/or the base material, for example.
The analysis reagent of the present invention may be, for example, an analysis kit. In this case, for example, the analysis kit may contain the nucleic acid sensor and the above-mentioned component(s), and they may be contained individually. The analysis kit may further contain the instruction manuals thereof, for example.
EXAMPLES
Example 1
A single-stranded nucleic acid element (II) including an AMP aptamer as a binding nucleic acid molecule (A) and EAD2 as a catalyst nucleic acid molecule (D) was produced, and the performance thereof as a nucleic acid sensor was checked.
As the nucleic acid elements, DNAs having the following respective sequences were synthesized (see FIGS. 2A and 2B). In each of the sequences, from the 5′ side thereof toward the 3′ side thereof, the sequence indicated by lower-case letters indicates a stem-forming sequence (SA), the poly dT indicated by capital letters indicates a loop-forming sequence (L2), the underlined portion on the 5′ side indicates EAD2 (D) of SEQ ID NO: 11, the sequence indicated by lower-case letters indicates a stem-forming sequence (SD), the poly dT indicated by capital letters indicates a loop-forming sequence (L1), and the underlined portion on the 3′ side indicates an AMP aptamer (A) of SEQ ID NO: 1.







AMP.D4.A4


(SEQ ID NO: 46)


5′-caggTTTCTGGGAGGGAGGGAGGGAccagTTTCCTGGGGGAGTATT


 


GCGGAGGAAGG-3′


 


AMP.D4.A5


(SEQ ID NO: 47)


5′-ccaggTTTCTGGGAGGGAGGGAGGGAccagTTTCCTGGGGGAGTAT


 


TGCGGAGGAAGG-3′


 


AMP.D5.A4


(SEQ ID NO: 50)


5′-caggTTTCTGGGAGGGAGGGAGGGAcccagTTTCCTGGGGGAGTAT


 


TGCGGAGGAAGG-3′






In Eppendorf tubes, 100 μL, each of the respective reaction solutions having the following composition was prepared and then caused to react for 60 seconds at 25° C. Thereafter, the absorbance (wavelength: 415 nm) of each reaction solution was measured. In the measurement, an absorbance measurement device (trade name: TECAN infinite, manufactured by TECAN) was used. In the reaction solution, the composition of the DNAzyme buffer contains 50 mmol/L Tris-HCl (pH7.4), 20 mmol/L KCl, and 0.05% TritonX-100. As a substrate, ABTS (2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic Acid Ammonium Salt) was used.







TABLE 1





(Composition of reaction solution)


















1 μmol/L
Nucleic acid sensor



3 μmol/L
Hemin



 1 mmol/L
AMP



50 mmol/L
DNAzyme buffer



 1 mmol/L
Substrate



0.5 mmol/L 
H2O2










In parallel, as a negative control, a reaction solution (NC) containing no nucleic acid sensor was subjected to the same reaction as described above. Moreover, as a positive control, a reaction solution (PC) using EAD2 of SEQ ID NO: 11 as a substitute for the nucleic acid sensor was subjected to the same reaction as described above.
EAD2(SEQ ID NO: 11) CTGGGAGGGAGGGAGGGA
These results are shown in FIG. 5. FIG. 5 is a graph showing absorbance of each of the reaction solutions. As shown in FIG. 5, in the result in the case of using the nucleic acid sensor of Example 1, the absorbance of the reaction solution containing 1 mmol/L AMP showed a significant difference to the absorbance of the reaction solution containing no AMP. As can be seen from this result, it can be said that, according to the nucleic acid sensor of Example 1, the presence or absence of and the concentration of AMP can be measured by measuring absorbance, and specifically, AMP can be detected even in the case of 1 mmol/L AMP.
Example 2
A measurement was performed using a nucleic acid element, AMP.D4.A5 (SEQ ID NO: 47), in Example 1 as a nucleic acid sensor with a change in concentration of AMP.
A reaction was performed in the same manner as in Example 1 except that the concentrations of AMP in each reaction solution were set to the predetermined concentrations (0, 50, and 500 μmol/L and 1, 2.5, and 5 mmol/L), and the reaction time was 170 seconds, and the absorbance was measured. A negative control (NC) and a positive control (PC) were the same as in Example 1 except for the concentrations of AMP.
These results are shown in FIG. 6. FIG. 6 is a graph showing absorbance of each of the reaction solutions. Five bars of each of NC, AMP.D4.A5, and PC in FIG. 6 show, from the left, the respective results of the cases where the concentrations of AMP were 0, 50, and 500 μmol/L and 1, 2.5, and 5 mmol/L. As shown in FIG. 6, in the negative control (NC) using only EAD2 that is DNAzyme, an increase in absorbance dependent on the concentration of AMP was not observed. In contrast, in the case of using the nucleic acid sensor of Example 2, an increase in absorbance dependent on the concentration of AMP was observed. As can be seen from this result, it can be said that, according to the nucleic acid sensor of Example 2, the concentration of AMP can be measured by measuring absorbance.
Example 3
A single-stranded nucleic acid element (II) including an AMP aptamer as a binding nucleic acid molecule (A) and neco0584 as a catalyst nucleic acid molecule (D) was produced, and the performance thereof as a nucleic acid sensor was checked.
As the nucleic acid elements, DNAs having the following respective sequences were synthesized (see FIGS. 2A and 2B). In each of the sequences, from the 5′ side thereof toward the 3′ side thereof, the sequence indicated by lower-case letters indicates a stem-forming sequence (SA), the poly dT indicated by capital letters indicates a loop-forming sequence (L2), the underlined portion on the 5′ side indicates neco0584 (D) of SEQ ID NO: 18, the sequence indicated by lower-case letters indicates a stem-forming sequence (SD), the poly dT indicated by capital letters indicates a loop-forming sequence (L1), and the underlined portion on the 3′ side indicates an ATP/AMP aptamer (A) of SEQ ID NO: 1.







AMP.neco.D3.A3


(SEQ ID NO: 53)


5′-aggTTTGGGTGGGAGGGTCGGGcccTTTCCTGGGGGAGTATTGCGG


 


AGGAAGG-3′


 


AMP.neco.D3.A4


(SEQ ID NO: 54)


5′-caggTTTGGGTGGGAGGGTCGGGcccTTTCCTGGGGGAGTATTGCG


 


GAGGAAGG-3′


 


AMP.neco.D3.A5


(SEQ ID NO: 55)


5′-ccaggTTTGGGTGGGAGGGTCGGGcccTTTCCTGGGGGAGTATTGC


 


GGAGGAAGG-3′


 


AMP.neco.D5.A5


(SEQ ID NO: 63)


5′-ccaggTTTGGGTGGGAGGGTCGGGcacccTTTCCTGGGGGAGTATT


 


GCGGAGGAAGG-3′






A reaction was performed in the same manner as in Examples 1 and 2 except that the above-described nucleic acid sensor was used, and the absorbance was measured. In parallel, as a negative control, a reaction solution (NC) containing no nucleic acid sensor was subjected to the same reaction as described above. Moreover, as a positive control, a reaction solution (PC) using neco0548 of SEQ ID NO: 18 as a substitute for the nucleic acid sensor was subjected to the same reaction as described above.
neco0584(SEQ ID NO: 18) GGGTGGGAGGGTCGGG
These results are shown in FIG. 7. FIG. 7 is a graph showing absorbance of each of the reaction solutions. As shown in FIG. 7, in the result in the case of using the nucleic acid sensor of Example 3, the absorbance of the reaction solution containing 1 mmol/L AMP showed a significant difference to the absorbance of the reaction solution containing no AMP. As can be seen from this result, it can be said that, according to the nucleic acid sensor of Example 3, the presence or absence of and the concentration of AMP can be measured by measuring absorbance, and specifically, AMP can be detected even in the case of 1 mmol/L AMP.
Example 4
A double-stranded nucleic acid element (I) including an ATP/AMP aptamer as a binding nucleic acid molecule (A) and DNAzyme as a catalyst nucleic acid molecule (D) was produced, and the performance thereof as a nucleic acid sensor was checked.
As a first strand nucleic acid (ss1), DNA having the following sequence was synthesized (see FIGS. 1A and 1B). In the following sequence, the underlined portion on the 5′ side indicates an AMP aptamer (A) of SEQ ID NO: 1, the poly dT indicates a loop-forming sequence (L1), and the underlined portion on the 3′ side indicates DNAzyme (D) of SEQ ID NO: 18 (neco0584).







AMP.neco.D0.A0


(SEQ ID NO: 2)


5′-CCTGGGGGAGTATTGCGGAGGAAGGTTTTTTTTGGGTGGGAGGGTC


 


GGG-3′






As a second strand (ss2) to be paired with the first strand (ss1), DNA having the following sequence was synthesized (see FIGS. 1A and 1B). In the following sequence, the underlined portion on the 5′ side indicates a stem-forming sequence (SD in each of FIGS. 1A and 1B) complementary to a 5′ side region of the DNAzyme of the first strand (ss1), the poly dT indicates a loop-forming sequence (L2 in each of FIGS. 1A and 1B), and the underlined portion on the 3′ side indicates a stem-forming sequence (SA in each of FIGS. 1A and 1B) complementary to a 3′ side region of an AMP aptamer of the first strand (ss1).









AMP.neco.D7.A8



(SEQ ID NO: 35)



5′-CTCCTTCCTTTTTTTTCCCACCC-3′






In Eppendorf tubes, 100 μL, each of the respective reaction solutions having the following composition was prepared and then caused to react for 60 seconds at 25° C. Thereafter, the absorbance (wavelength: 415 nm) of each reaction solution was measured. In the measurement, an absorbance measurement device (trade name: TECAN infinite, manufactured by TECAN) was used. In the reaction solution, the composition of the DNAzyme buffer contains 50 mmol/L Tris-HCl (pH7.4), 20 mmol/L KCl, 150 mmol/L NaCl, and 0.05% TritonX-100. As a substrate, ABTS was used.







TABLE 2





(Composition of reaction solution)


















1 μmol/L
First strand (ss1)



2 μmol/L
Second strand (ss2)



3 μmol/L
Hemin



 1 mmol/L
AMP



50 mmol/L
DNAzyme buffer



 1 mmol/L
Substrate



0.5 mmol/L 
H2O2










In parallel, as a positive control, a reaction solution (PC) using DNAzyme of SEQ ID NO: 18 (neco0584) as a substitute for the nucleic acid sensor was subjected to the same reaction as described above. Moreover, as a negative control, a reaction solution (NC) containing no nucleic acid sensor was subjected to the same reaction as described above.
neco0584 (SEQ ID NO: 18) GGGTGGGAGGGTCGGG
These results are shown in FIG. 8. FIG. 8 is a graph showing absorbance of each of the reaction solutions. As shown in FIG. 8, in the result in the case of using the nucleic acid sensor of Example 4, the absorbance of the reaction solution containing 1 mmol/L AMP showed a significant difference to the absorbance of the reaction solution containing no AMP. As can be seen from this result, it can be said that, according to the nucleic acid sensor of Example 4, the presence or absence of and the concentration of AMP can be measured by measuring absorbance, and specifically, AMP can be detected even in the case of 1 μmol/L AMP.
While the invention has been particularly shown and described with reference to exemplary embodiments thereof, the invention is not limited to these embodiments. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the claims.
While the invention has been particularly shown and described with reference to exemplary embodiments thereof, the invention is not limited to these embodiments. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the claims.
This application is based upon and claims the benefit of priority from International Patent Application PCT/JP2012/57635, filed on Mar. 23, 2012, the disclosure of which is incorporated herein its entirety by reference.
INDUSTRIAL APPLICABILITY
According to the present invention, the catalytic function of the catalyst nucleic acid molecule (D) can be switched ON/OFF by a binding/non-binding of the binding nucleic acid molecule (A) with a target, and the sensitivity thereof is superior. Therefore, the presence or absence of and the amount of the target can be easily detected by detecting the catalytic function of the catalyst nucleic acid molecule. Moreover, the analysis device of the present invention uses the nucleic acid sensor as mentioned above. Therefore, for example, the analysis device can be downsized and formed into a chip, and many specimens can be easily analyzed by the analysis device. Thus, it can be said that the present invention is a really useful technology for researches and inspections in various fields such as clinical medical care, food, and environment, for example.

## Claims

1. A nucleic acid sensor comprising at least one nucleic acid element chosen from (I) and (II) that comprises a catalyst nucleic acid molecule (D) and a binding nucleic acid molecule (A),
(I) a single-stranded nucleic acid element comprising the binding nucleic acid molecule (A), a loop-forming sequence (L1), a stem-forming sequence (SD), the catalyst nucleic acid molecule (D), a loop-forming sequence (L2), and a stem-forming sequence (SA) linked in this order,
wherein
a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side is complementary to the stem-forming sequence (SA),
a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side is complementary to the stem-forming sequence (SD),
the loop-forming sequence (L1) is non-complementary to the loop-forming sequence (L2), and
wherein,
in the absence of a target, the catalyst nucleic acid molecule (D) is caged by stem formation in each of the stem-forming sequences (SA) and (SD),
in the presence of a target, the stem-forming sequence (SA) and the stem-forming sequence (SD) are released from the binding nucleic acid molecule (A) and the catalyst nucleic acid molecule (D), and the catalyst nucleic acid molecule (D) is released,
a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side and the stem-forming sequence (SA) form a stem,
a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side and the stem-forming sequence (SD) form a stem, and
the loop-forming sequences (L1) and (L2) form an internal loop between the two stems;


(II) a single-stranded nucleic acid element comprising the catalyst nucleic acid molecule (D), a loop-forming sequence (L2), a stem-forming sequence (SA), the binding nucleic acid molecule (A), a loop-forming sequence (L1), and a stem-forming sequence (SD) linked in this order,
wherein
a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side is complementary to the stem-forming sequence (SD),
a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side is complementary to the stem-forming sequence (SA),
the loop-forming sequence (L1) is non-complementary to the loop-forming sequence (L2), and
wherein,
in the absence of a target, the catalyst nucleic acid molecule (D) is caged by stem formation in each of the stem-forming sequences (SA) and (SD),
in the presence of a target, the stem-forming sequence (SA) and the stem-forming sequence (SD) are released from the binding nucleic acid molecule (A) and the catalyst nucleic acid molecule (D), and the catalyst nucleic acid molecule (D) is released,
a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side and the stem-forming sequence (SA) form a stem,
a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side and the stem-forming sequence (SD) form a stem, and
the loop-forming sequences (L1) and (L2) form an internal loop between the two stems,


further wherein the catalyst nucleic acid molecule (D) exerts an oxidation-reduction reaction and the target is ATP or AMP.

2. The nucleic acid sensor according to claim 1, wherein
the nucleic acid element (I) comprises, from the 3′ side thereof, the binding nucleic acid molecule (A), the loop-forming sequence (L1), the stem-forming sequence (SD), the catalyst nucleic acid molecule (D), the loop-forming sequence (L2), and the stem-forming sequence (SA) linked in this order,
a 5′ terminal region of the binding nucleic acid molecule (A) is complementary to the stem-forming sequence (SA), and
a 5 terminal region of the catalyst nucleic acid molecule (D) is complementary to the stem-forming sequence (SD).

3. The nucleic acid sensor according to claim 2, wherein
the length of each of the loop-forming sequences (L1) and (L2) ranges from 1- to 30-nucleotides.

4. The nucleic acid sensor according to claim 2, wherein
the length of the stem-forming sequence (SA) ranges from 1- to 60-nucleotides, and
the length of the stem-forming sequence (SD) ranges from 1- to 30-nucleotides.

5. The nucleic acid sensor according to claim 1, wherein
the length of the binding nucleic acid molecule (A) ranges from 18- to 60-nucleotides.

6. The nucleic acid sensor according to claim 1, wherein
the binding nucleic acid molecule (A) comprises the following polynucleotide (a1), (a2), (a3), or (a4):
(a1) a polynucleotide composed of a base sequence of SEQ ID NO: 1,
(a2) a polynucleotide that is composed of a base sequence obtained by substitution, deletion, addition and/or insertion of at least one base in the base sequence of the polynucleotide (a1),
(a3) a polynucleotide that is composed of a base sequence with 50% or more identity with the base sequence of the polynucleotide (a1), and
(a4) a polynucleotide that is composed of a base sequence complementary to a base sequence that hybridizes to the base sequence of the polynucleotide (a1) under stringent conditions.

7. The nucleic acid sensor according to claim 1, wherein
the length of the catalyst nucleic acid molecule (D) ranges from 15- to 30-nucleotides.

8. The nucleic acid sensor according to claim 1, wherein
the catalyst nucleic acid molecule (D) comprises the following polynucleotide (d1), (d2), (d3), or (d4):
(d1) a polynucleotide composed of a base sequence of any of SEQ ID NOs: 11 to 31 and 66 to 85,
(d2) a polynucleotide that is composed of a base sequence obtained by substitution, deletion, addition, and/or insertion of at least one base in the base sequence of the polynucleotide (d1) that exerts the oxidation-reduction reaction,
(d3) a polynucleotide that is composed of a base sequence with 50% or more identity with the base sequence of the polynucleotide (d1) that exerts the oxidation-reduction reaction, and
(d4) a polynucleotide that is composed of a base sequence complementary to a base sequence that hybridizes to the base sequence of the polynucleotide (d1) under stringent conditions that exerts the oxidation-reduction reaction.

9. A device for analyzing a target, comprising:
a base material;
a nucleic acid sensor; and
a detection part, wherein
the nucleic acid sensor and the detection part are disposed on the base material,
the nucleic acid sensor is the nucleic acid sensor according to claim 1, and
the detection part is a detection part that detects when the catalyst nucleic acid molecule (D) in the nucleic acid sensor is released.

10. The device according to claim 9, wherein the nucleic acid sensor is linked with the base material via a linker.

11. The device according to claim 9, wherein the nucleic acid sensor is disposed on the detection part.

12. The device according to claim 9, wherein the detection part detects a signal generated when the catalyst nucleic acid molecule (D) is released.

13. The device according to claim 12, wherein the signal is an optical signal or an electrochemical signal.

14. The device according to claim 9, further comprising:
a reagent part, wherein the reagent part comprises a substrate that reacts with the catalyst nucleic acid molecule (D).

15. A method for analyzing a target, comprising:
a contact step of causing a sample to be in contact with the nucleic acid sensor according to claim 1; and
a detection step of detecting that the catalyst nucleic acid molecule (D) in the nucleic acid sensor is released.

16. The method according to claim 15, wherein the detection step is performed in the presence of a substrate that reacts with the catalyst nucleic acid molecule (D).

17. A method for analyzing a target, comprising:
a contact step of causing a sample to be in contact with the device according to claim 9; and
a detection step of detecting that the catalyst nucleic acid molecule (D) in the detection part of the device is released.

18. The method according to claim 17, wherein the detection step is performed in the presence of a substrate that reacts with the catalyst nucleic acid molecule (D).