Patent Grant
9637737
The present invention relates to a method for evaluating redox activity of a nucleic acid molecule and a nucleic acid molecule having redox activity.
It is required to detect a target in various fields such as clinical medical care, food, and environment. In the detection of a target, interactions with the target are generally utilized, and among them, a method using an antibody that specifically binds to a target is widely used. In this method, for example, a target is bound to an antibody labeled with oxidoreductase such as peroxidase. Then, a chromogenic reaction is conducted by the enzyme in the labeled antibody using a chromogenic substrate, and the development in color is detected. By the detection of the development in color, analyses of the target such as qualitative analysis and quantitative analysis are performed indirectly.
However, since the antibody is obtained by immunizing animals, it is really difficult to obtain an antigen specific to a toxic target or a low-molecular-weight target. Hence, recently, a nucleic acid that binds to a target, i.e., a nucleic acid aptamer (hereinafter, merely referred to as an aptamer) has been focused on. The aptamer can be obtained in a test tube. Therefore, for example, it is possible to obtain aptamers to a toxic target and a low-molecular-weight target. Further, it has been attempted to use such aptamer as substitute for the antibody in detection of a target in combination with DNAzyme exerting catalytic activity as in peroxidase. The DNAzyme generally is DNA that exerts a catalytic property of peroxidase by having a guanine-rich structural motif, having a G-quadruplex structure, and forming a complex by binding to hemin.
In the detection of a target, a single-stranded nucleic acid element obtained by linking a single-stranded aptamer and a single-stranded DNAzyme is specifically utilized (Non-Patent Document 1). The single-stranded nucleic acid element forms a stem structure by self-annealing in the absence of a target, and the DNAzyme cannot form G-quadruplex by the stem structure. Therefore, the DNAzyme in the single-stranded nucleic acid element cannot bind to hemin and cannot achieve a catalytic property in the absence of a target. On the other hand, in the presence of a target, the single-stranded nucleic acid element releases the stem structure by binding the target to the aptamer. Therefore, in the presence of a target, the DNAzyme in the single-stranded nucleic acid element forms G-quadruplex and exerts the catalytic property by binding to hemin. Thus, when a chromogenic substrate to redox activity is present together, a chromogenic reaction occurs in the presence of the target, and a chromogenic reaction does not occur in the absence of the target. Therefore, it becomes possible to analyze the target by detecting the chromogenic reaction. Furthermore, it is not required to label the target, and thus, it becomes possible to directly detect various targets including a low-molecular-weight target.
As described above, the single-stranded nucleic acid element is required to control activity of the DNAzyme by a conformation of the aptamer. Therefore, for example, it is desired to combine DNAzyme whose activity can be easily controlled according to the sequence of an aptamer to be used.
However, there is only a limited number of DNAzymes that have been reported. Therefore, when a combination with a predetermined aptamer is decided, there has no choice but to select from the limited number of DNAzymes. Thus, there is a limitation in structuring a nucleic acid element with superior accuracy according to the target. Moreover, in order to make it possible to perform detection with superior sensitivity, DNAzyme which highly exerts redox activity is required.
Therefore, it has been attempted to obtain novel DNAzyme. However, in screening of DNAzyme, there has no choice but to determine each of activities of candidate nucleic acid molecules, and the operation thereof is really complicated.
Hence, the present invention is intended to provide a novel technique by which redox activity of a nucleic acid molecule can be easily evaluated.
The evaluation method according to the present invention is a method for evaluating redox activity of a nucleic acid molecule, the method including: a detection step of electrochemically detecting a redox reaction to a substrate, the redox reaction being catalyzed by at least one nucleic acid molecule to be evaluated, using a device that electrochemically detects a redox reaction; and an evaluation step of evaluating redox activity of the at least one nucleic acid molecule from a result of the detection of the redox reaction, wherein the device includes a base provided with a detection portion, the detection portion includes an electrode system, and the nucleic acid molecule to be evaluated is arranged on the base.
The nucleic acid molecule according to the present invention is a nucleic acid molecule having redox activity, containing at least one polynucleotide selected from the group consisting of SEQ ID NOs: 1 to 132.
According to the evaluation method of the present invention, the presence or absence of redox activity of a nucleic acid molecule to be evaluated and the intensity of the redox activity can be evaluated easily. Moreover, according to the evaluation method of the present invention, a plurality of kinds of nucleic acid molecules can be evaluated simultaneously, for example. Therefore, an intended nucleic acid molecule can be effectively screened. The nucleic acid molecule having redox activity is, for example, as mentioned above, used as substitute for an enzyme such as peroxidase and thus is useful in various fields such as clinical medical care, food, and environment.
The evaluation method according to the present invention is, as mentioned above, a method for evaluating redox activity of a nucleic acid molecule, the method including: a detection step of electrochemically detecting a redox reaction to a substrate, the redox reaction being catalyzed by at least one nucleic acid molecule to be evaluated, using a device that electrochemically detects a redox reaction; and an evaluation step of evaluating redox activity of the at least one nucleic acid molecule from a result of the detection of the redox reaction, wherein the device includes a base provided with a detection portion, the detection portion includes an electrode system, and the nucleic acid molecule to be evaluated is arranged on the base.
Only one kind of the nucleic acid molecule to be evaluated may be used, and it is preferred that the nucleic acid molecule to be evaluated includes a plurality of kinds of nucleic acid molecules, for example. In the latter case, for example, by arranging a plurality of kinds of nucleic acid molecules on the base, the plurality of kinds of nucleic acid molecules can be evaluated using the single base.
The nucleic acid molecule to be evaluated is, for example, a molecule containing a nucleotide residue. The nucleic acid molecule may be, for example, a molecule composed of only a nucleotide residue or a molecule containing a nucleotide residue. Examples of the nucleotide include ribonucleotide, deoxyribonucleotide, and derivatives thereof. The nucleic acid molecule may contain any one kind of, two or more kinds of, or all of ribonucleotide, deoxyribonucleotide, and derivatives thereof. Specifically, the nucleic acid molecule may be, for example, RNA containing ribonucleotide and/or a derivative thereof, DNA containing deoxyribonucleotide and/or a derivative thereof, or a chimera (DNA/RNA) containing the former and the latter. The nucleic acid molecule may be a single strand or a double strand and is preferably a single strand. When the nucleic acid molecule is a single strand, examples of the nucleic acid molecule include a single-stranded DNA, a single-stranded RNA, and a single-stranded chimera (DNA/RNA). When the nucleic acid molecule is a double strand, examples of the nucleic acid molecule include a double-stranded DNA, a double-stranded RNA, a DNA-RNA double strand, and a double-stranded chimera (DNA/RNA). The length of the nucleic acid molecule is not particularly limited and is, for example, from 11 to 80 bases.
The nucleotide may include, as bases, for example, natural bases (non-artificial bases) and non-natural bases (artificial bases). Examples of the natural bases include A, C, G, T, U, and modified bases thereof. Examples of the modification include methylation, fluorination, amination, and thiation. Examples of the non-natural bases 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 a 2′-methylated-uracil nucleotide residue, a 2′-methylated-cytosine nucleotide residue, a 2′-fluorinated-uracil nucleotide residue, a 2′-fluorinated-cytosine nucleotide residue, a 2′-aminated-uracil nucleotide residue, a 2′-aminated-cytosine nucleotide residue, a 2′-thiated-uracil nucleotide residue, and a 2′-thiated-cytosine nucleotide residue. Examples of the nucleic acid molecule include PNA (peptide nucleic acid) and LNA (Locked Nucleic Acid).
In the present invention, the redox reaction is only necessary to be a reaction in which a transfer of electrons between two substrates is generated in a process of generating a product from the substrates, for example. The kind of the redox reaction is not particularly limited. The activity that catalyzes the redox reaction can be, for example, activity that is the same as an enzyme, and can be, specifically, for example, activity that is the same as peroxidase (hereinafter also referred to as peroxidase-like activity). The peroxidase-like activity can be, for example, horseradish-derived peroxidase (HRP) activity. When the nucleic acid molecule to be evaluated is DNA and has the redox activity, the DNA can be referred to as DNA enzyme or DNAzyme, for example. When the nucleic acid molecule is RNA and has the redox activity, the RNA can be referred to as RNA enzyme or RNAzyme, for example.
In the present invention, the detection portion of the device is only necessary to detect an electrical signal that is generated by a redox reaction that is catalyzed by the nucleic acid molecule to be evaluated. The detection portion includes an electrode system as mentioned above. The electrode system may include a working electrode and a counter electrode or may include 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.
The detection portion can be formed by arranging the electrodes on the surface of the base, for example. A method for arranging the electrodes is not particularly limited, and a conventionally known method can be employed, for example. Specific examples of the method include thin-film forming methods such as an evaporation method, a sputtering method, a screen printing method, and a plating method. The electrodes may be arranged directly or indirectly on the base, for example. The indirect arrangement can be, for example, an arrangement via other members.
The base is not particularly limited and is, for example, preferably a base having an insulating surface. The base may be, for example, a base containing an insulating material or being composed of an insulating material or a base including an insulating layer that has a surface containing an insulating material or an insulating layer composed of the insulating material. The insulating material is not particularly limited, and examples thereof include conventionally known materials such as glass, ceramics, insulating plastics, 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.
As mentioned above, only one kind of the nucleic acid molecule to be evaluated may be used, it is preferred that the nucleic acid molecule to be evaluated includes a plurality of kinds of nucleic acid molecules, and specifically, the plurality of kinds of nucleic acid molecules is arranged on the base. The device is, for example, preferably a microarray obtained by arranging the plurality of nucleic acid molecules on the base. It is preferred that the plurality of kinds of nucleic acid molecules is arranged on the base in the matrix state, for example. In order to make it possible to detect a redox reaction of the nucleic acid molecule according to the kind thereof, it is preferred that the device has a plurality of detection portions, and a different kind of nucleic acid molecule is arranged in each detection portion, for example. Specifically, the device can be formed by fractionating the surface of the base into matrixes, forming an electrode system such as mentioned above in each fractional region to form detection portions, and arranging the nucleic acid molecule in each detection portion, for example. Moreover, a device obtained by binding the nucleic acid molecule to be evaluated to a probe on a commercially available electrochemical detection-type system microarray can be used as a device for evaluation. The commercially available microarray can be, for example, CombiMatrix ElectraSense microarray (trade name) (manufactured by CombiMatrix).
The nucleic acid molecule to be evaluated is only necessary to be arranged on the base as mentioned above and is preferably immobilized on the base. The nucleic acid molecule may be arranged directly or indirectly on the base, for example. Specifically, the nucleic acid molecule is, for example, preferably arranged on the detection portion in the base, more preferably arranged on any of the electrodes in the detection portion, and yet more preferably arranged on the working electrode among the electrodes. The nucleic acid molecule may be arranged directly or indirectly on the detection portion or any of the electrodes, for example. Hereinafter, “arrangement or immobilization of the nucleic acid molecule on the base” encompasses the meaning of arrangement or immobilization on the detection portion in the base or any of the electrodes in the detection portion, unless otherwise shown.
A method for arranging the nucleic acid molecule is not particularly limited, and a known nucleic acid immobilization method can be employed. The above-described immobilization method can be, for example, a method for immobilizing a previously provided nucleic acid molecule on the base, preferably on the detection portion, more preferably on any of the electrodes. This method is, for example, a method utilizing photolithography, and a specific example thereof is shown in U.S. Pat. No. 5,424,186 or the like. Furthermore, the immobilization method can be, for example, a method for synthesizing a nucleic acid on the base, preferably on the detection portion, more preferably on any of the electrodes. This method can be, for example, a spot method, and a specific example thereof is shown in U.S. Pat. No. 5,807,522, JP H10-503841, or the like.
The nucleic acid molecule may be immobilized or the base in any of the 5′ end side and the 3′ end side.
The nucleic acid molecule is preferably arranged on the base via a linker, for example. The linker preferably contains a nucleotide residue, for example. The linker may be composed of only a nucleotide residue or may contain a nucleotide residue, for example. The nucleotide is the same as mentioned above. The length of the linker is not particularly limited and is, for example, from 1 to 60 bases, preferably from 6 to 60 bases, more preferably from 20 to 30 bases. The linker is also referred to as a spacer, for example.
In the device, the nucleic acid molecule to be evaluated may be in the state of linking with an aptamer, for example. Hereinafter, an object obtained by linking between the nucleic acid molecule to be evaluated and the aptamer is referred to as a nucleic acid element. The aptamer is not particularly limited and is, for example, an aptamer capable of binding to a specific target. For example, it is preferred that the aptamer is a nucleic acid molecule containing a nucleotide residue as a component. The nucleotide is not particularly limited and is the same as mentioned above. For example, the aptamer capable of binding to the target can be produced by a conventionally known SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method or the like. The aptamer may be, for example, a single strand or a double strand and is preferably a single strand.
The nucleic acid molecule and the aptamer may be, for example, directly or indirectly bound to each other. In the latter case, they may be bound to each other via a linker, for example. For example, the 5′ end of one of the nucleic acid molecule and the aptamer may be linked with the 3′ end of the other, the 5′ end of the nucleic acid molecule may be linked with the 5′ end of the aptamer, or the 3′ end of the nucleic acid molecule may be linked with the 3′ end of the aptamer.
When the aptamer is linked with the nucleic acid molecule, it is preferred that one end of the nucleic acid molecule is bound to the base, and the other end is bound to the aptamer, for example. As mentioned above, the base can be bound to the nucleic acid molecule via a linker, and the nucleic acid molecule can be bound to the aptamer via a linker, for example. It is preferred that the linkage between the nucleic acid molecule and the aptamer is, for example, a linkage between a single stranded nucleic acid molecule and a single-stranded aptamer. The nucleic acid element obtained by linking between the nucleic acid molecule and the aptamer may form a stem structure and/or a loop structure by self-annealing, for example.
With respect to the evaluation method according to the present invention, a first embodiment shows, as an example, a method using a device in which the nucleic acid molecule to be evaluated is arranged, and a second embodiment shows, as an example, a method using a device in which the nucleic acid molecule binding to the aptamer is arranged, i.e., a device in which the nucleic acid element is arranged. The present invention is not limited to these embodiments.
In the first embodiment of the present invention, a device in which a nucleic acid molecule to be evaluated is arranged on the base is used as mentioned above.
First, in the detection step, for example, a redox reaction catalyzed by a nucleic acid molecule is electrochemically detected in the presence of a substrate. When the nucleic acid molecule has redox activity, a product is generated from the substrate by the nucleic acid molecule, for example, and in the process of the generation, electrons are transferred, for example. This electron transfer can be electrochemically detected as an electrical signal in a detection portion of the device by applying a voltage to electrodes, for example. The detection of the electrical signal can be performed by measuring intensity of the electrical signal such as a current, for example.
The substrate can be externally supplied to the nucleic acid molecule in the device in the detection step, 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, porphyrin can be present together besides the substrate, for example. Some of known DNAzymes exert superior redox activity by forming a complex with porphyrin, for example. Thus, in the present invention, for example, porphyrin may be caused to be present together to form a complex with the porphyrin and detect redox activity of the complex.
The porphyrin is not particularly limited, and examples thereof include unsubstituted porphyrin and a derivative thereof. The derivative can be, for example, metal porphyrin obtained by forming a complex of substituted 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(Fe3+) complex. The porphyrin is, for example, preferably the metal porphyrin, more preferably hemin.
In the detection step, conditions under which the redox reaction is performed are not particularly limited. The pH is, for example, from 7.4 to 9.0, preferably from 7.4 to 8.5, more preferably 7.4. It is preferred that the pH is adjusted using a buffer solution, for example, and a buffer solution such as Tris-HCl having the above-mentioned pH can be used, for example. The substrate is preferably added to the nucleic acid molecule as a substrate solution by mixing with a buffer solution, for example. The concentration of the substrate in the substrate solution is not particularly limited and is, for example, from 10 to 200 mmol/L, preferably from 20 to 100 mmol/L, more preferably from 40 to 60 mmol/L, particularly preferably 50 mmol/L.
Then, based on a result of the detection in the detection step, redox activity of the nucleic acid molecule is evaluated in an evaluation step. In the evaluation step, the presence or absence of redox activity or the intensity of the redox activity may be evaluated, for example. In the latter case, for example, relative intensity of the activity can be evaluated relative to redox activity of any nucleic acid molecule, and the any nucleic acid molecule preferably has redox activity.
In the second embodiment of the present invention, as mentioned above, a device in which a nucleic acid molecule to be evaluated bound to an aptamer is arranged on a base is used. The second embodiment is the same as the first embodiment unless otherwise indicated.
First, in the detection step, for example, a redox reaction catalyzed by a nucleic acid molecule is electrochemically detected in the presence of a substrate and a target. It is particularly preferred that the detection step includes a step of detecting a redox reaction in the presence of the substrate and the in the absence of the target and a step of detecting a redox reaction in the presence of the substrate and the target. Any of the detection in the absence of a target and the detection in the presence of a target may be performed prior to the other. For example, it is preferred that the detection in the absence of a target is performed prior to the other because the target binding to an aptamer is not required to be released.
The reason why the detection in the absence of a target and the detection in the presence of a target are performed is as follows. In the case where an aptamer capable of binding to a target and a nucleic acid molecule having redox activity are used in detection of a target, it is desired that the nucleic acid molecule having redox activity exerts activity only when the target is present and does not exert activity when the target is absent. This is because, when activity is exerted in the absence of a target, a false-positive result is obtained in the detection of the target. Therefore, the presence or absence of redox activity in the absence of a target and the presence or absence of redox activity in the presence of a target are important for establishing an electrochemical method in the detection of a target using an aptamer.
It is considered that the following structural relationship is satisfied between the nucleic acid molecule and the aptamer in the case where the nucleic acid molecule does not exert activity in the absence of a target and exerts activity in the presence of a target, for example. The aptamer generally changes its conformation by recognizing and binding to a target. Therefore, by a conformation of an aptamer in the absence of a target, the nucleic acid molecule binding to the aptamer has a structure in which activity is switched off. On the other hand, by a conformation of the aptamer changed by binding to the target, suppression of activity of the nucleic acid molecule is released, so that the activity of the nucleic acid molecule is switched on only in the case where a target is present.
In the detection step, the substrate and the target can be externally supplied to the nucleic acid molecule in the device, for example. The order of adding the substrate and the target is not particularly limited. For example, it is possible that one of them is added, and the other is thereafter added, or both of them are added at the same time. Moreover, the porphyrin may be present together as in the first embodiment.
The substrate is not particularly limited and is the same as mentioned above. The target and the aptamer are not particularly limited, and a desired target and an aptamer capable of binding thereto can be used.
Then, in the evaluation step, based on a result of the detection in the detection step, redox activity of the nucleic acid molecule is evaluated. In the evaluation step, the presence or absence of redox activity or the intensity of the redox activity may be evaluated, for example. In the latter case, for example, relative intensity of the activity can be evaluated relative to redox activity of any nucleic acid molecule, and the any nucleic acid molecule preferably has redox activity.
It is preferred that in the evaluation step, redox activity of a nucleic acid molecule that does not exert redox activity in the absence of a target is evaluated in the presence of a target, for example. In the evaluation step, the presence or absence of redox activity or the intensity of redox activity may be evaluated, for example. In the latter case, for example, relative intensity of the activity can be evaluated relative to redox activity of any nucleic acid molecule.
The screening method according to the present invention is, as mentioned above, a method for screening a nucleic acid molecule having redox activity, including: evaluating redox activity of at least one nucleic acid molecule to be evaluated by the method according to the present invention using a device; and screening a nucleic acid molecule having redox activity.
The screening method according to the present invention is characterized in that redox activity of the nucleic acid molecule to be evaluated is evaluated by the evaluation method according to the present invention, and other steps and conditions are not at all limited.
In the present invention, based on a result of the evaluation of redox activity, a nucleic acid molecule exerting redox activity can be selected, and furthermore, a nucleic acid molecule exerting relatively intense redox activity can be selected as an intended nucleic acid molecule, for example. Moreover, as mentioned above, for example, a nucleic acid molecule that does not exert activity in the absence of a target and exerts activity in the presence of a target can be selected as an intended nucleic acid molecule.
The nucleic acid molecule having redox activity according to the present invention contains at least one polynucleotide selected from the group consisting of the following (a) to (d):
(a) a polynucleotide composed of any of base sequences of SEQ ID NOs: 1 to 132;
(b) a polynucleotide being composed of a base sequence obtained by deletion, substitution, insertion and/or addition of one or more bases in any of the base sequences in the polynucleotide (a) and having redox activity;
(c) a polynucleotide being composed of a base sequence having an identity of 80% or more to any of the base sequences in the polynucleotide (a) and having redox activity; and
(d) a polynucleotide being composed of a base sequence complementary to a polynucleotide that can hybridize with the polynucleotide (a) under stringent conditions and having redox activity.
The nucleic acid molecule according to the present invention may be, for example, a molecule being composed of or containing any of the polynucleotides (a) to (d). In the latter case, the nucleic acid molecule according to the present invention may contain two or more of the polynucleotides (a) to (d) as described below, for example. The two or more of the polynucleotides may be the same sequences or different sequences. In the latter case, the nucleic acid molecule according to the present invention may further have a linker and/or an additional sequence, for example. The nucleic acid molecule according to the present invention is also referred to as DNAzyme.
The polynucleotide (a) is a polynucleotide composed of any of base sequences of SEQ ID NOs: 1 to 132.
In the polynucleotide (b), “one or more” is not limited as long as the polynucleotide (b) is in a range of having redox activity, for example. The “one or more” is, for example, from 1 to 5, preferably from 1 to 3, more preferably from 1 or 2 in any of the base sequences in the polynucleotide (a).
In the polynucleotide (c), “an identity” is not limited as long as the polynucleotide (c) is in a range of having redox activity, for example. The identity is, for example, 80% or more, 85% or more, preferably 90% or more, more preferably 95% or more, 96% or more, 97% or more, yet more preferably 98% or more, particularly preferably 99% or more. The identity can be calculated by an analysis software program such as BLAST or FASTA using default parameters, for example (the same applies hereinafter).
In the polynucleotide (d), “a polynucleotide that can hybridize” is, for example, a polynucleotide completely or partially complementary to the polynucleotide (a). The hybridization can be detected by any of various hybridization assays, for example. The hybridization assays are not particularly limited, and for example, a method described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press (1989) or the like can be employed.
In the polynucleotide (d), the “stringent conditions” may be, for example, any of low stringent conditions, middle stringent conditions, and high stringent conditions. The “low stringent conditions” are, for example, conditions of 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, and 32° C. The “middle stringent conditions” are, for example, conditions of 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, and 42° C. The “high stringent conditions” are, for example, conditions of 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, and 50° C. Those skilled in the art can set the extent of stringency by appropriately selecting conditions such the temperature, the salt concentration, the concentration and the length of a probe, the ionic strength, and the time, for example. As the “stringent conditions”, conditions described in Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press (1989) can be employed, for example.
A component of the polynucleotide is, for example, a nucleotide residue. Examples of the nucleic acid molecule according to the present invention include DNA composed of only a deoxyribonucleotide residue and DNA containing one or more ribonucleotide residues. In the latter case, the one or more is not particularly limited and is, for example, from 1 to 3, preferably 1 or 2 in the polynucleotide.
In the nucleic acid molecule according to the present invention, the polynucleotide is preferably a single-stranded polynucleotide. It is preferred that the single-stranded polynucleotide is capable of forming a stem structure and a loop structure by self-annealing, for example. It is preferred that the polynucleotide is capable of forming a stem-loop structure, an internal loop structure, and/or a bulge structure, for example.
The nucleic acid molecule according to the present invention may be, for example, a double-stranded polynucleotide. When the nucleic acid molecule according to the present invention is a double-stranded polynucleotide, one of single-stranded polynucleotides of the double-stranded polynucleotide is any of the above-described polynucleotides, and the other single-stranded polynucleotide is not limited, for example. The other single-stranded polynucleotide can be, for example, a polynucleotide composed of a base sequence complementary to any of the above-described polynucleotides. When the nucleic acid molecule according to the present invention is a double-stranded polynucleotide, it is preferred that the double-stranded polynucleotide is dissociated into single-stranded polynucleotides by denaturation or the like prior to the use thereof, for example. The dissociated single-stranded polynucleotides may form a stem structure and a loop structure as mentioned above, for example.
In the present invention, “being capable of forming a stem structure and a loop structure” includes: actually forming a stem structure and a loop structure; and being capable of forming a stem structure and a loop structure even if they are not formed, for example. “Bing capable of forming a stem structure and a loop structure” includes the case where the formation is checked experimentally and the case where the formation is predicted by a simulation using a computer or the like, for example.
The nucleic acid molecule according to the present invention has redox activity, so that it can be used as a substitute for oxidoreductase and is applicable to detection of a target using an aptamer such as mentioned above, for example.
The conditions under which a redox reaction of a nucleic acid molecule to be evaluated was detected were considered.
(1) Immobilization of Polynucleotide
A known polynucleotide was immobilized, via a spacer, on an electrode of a commercially available electrochemical detection-type microarray (trade name: CombiMatrix ElectraSense microarray, manufactured by CombiMatrix) as shown below.
In the known polynucleotide, EAD2 (SEQ ID NO: 133) (see Cheng, X., et al., (2009), Biochemistry, 48, 7817-7823) was used as DNAzyme. As a negative control, DNA aptamer SA (SEQ ID NO: 134) that binds to streptavidin was used.
The lengths of the spacer were 0-mer (no spacer), 8-mer, 16-mer, and 24-mer. The sequence of the spacer was poly dT.
The immobilization was performed by binding the 3′ end of the spacer with one electrode of the microarray and binding the 3′ end of the known polynucleotide to the 5′ end of the spacer. 250 each of four kinds of EAD2s (dT0, dT8, dT16, dT24) having different lengths of the spacer and four kinds of SAs (dT0, dT8, dT16, dT24) having different lengths of the spacer were randomly immobilized on the microarray.
(2) Detection of Redox Reaction
Hydrogen peroxide was added to the microarray as a substrate, and an electrical signal generated by a redox reaction was measured as a current. In the measurement, a measurement device (product name: ElectraSense Reader, manufactured by CombiMatrix) was used (hereinafter the same). Hydrogen peroxide was added to various buffer solutions so as to have predetermined concentrations (0, 1, 2, 4, 8 mmol/L), and the resultant solutions were added as substrate solutions. As the buffer solutions, a tris buffer solution (pH7.4), a tris buffer solution (pH8.0), a tris buffer solution (pH8.5), and a tris buffer solution (pH9.0) were used, and the pHs of the resultant solutions were adjusted so as to have each pH in parentheses after the addition of hydrogen peroxide.
Results of these are shown in
Redox reactions of 250 each of four kinds of EAD2s (dT0, dT8, dT16, dT24) having different lengths of the spacer were measured under the same conditions as in “A. Consideration of conditions”.
Results of these are shown in
Whether or not the results obtained by the measurements of 250 each of four kinds of EAD2s (dT0, dT8, dT16, dT24) having different lengths of the spacer and the results obtained by the measurements of 250 each of four kinds of SAs (dT0, dT8, dT16, dT24) having different lengths of the spacer in “A. Consideration of conditions” were normally-distributed was checked. In addition, a P value was determined by a Kolmoforov-Smirnov test.
Results of these are shown in
A plurality of polynucleotides composed of different sequences were immobilized on a microarray chip, and redox reactions thereof were measured.
As shown below, a plurality of polynucleotides were immobilized, via a spacer composed of 24-mer poly dT, on an electrode of a commercially available microarray chip (trade name: CustomArray (registered trademark) 12K, manufactured by CombiMatrix).
In the polynucleotides, as DNAzyme, EAD2 (SEQ ID NO: 133), an aptamer SA (SEQ ID NO: 134), c-Myc (SEQ ID NO: 135) that is a partial sequence in a promoter region of a transcription factor c-Myc gene, and TA (SEQ ID NO: 136) that is an aptamer to thrombin were used.
Moreover, modified polynucleotides obtained by modifying sequences of the EAD2 (SEQ ID NO: 133), the c-Myc (SEQ ID NO: 135), and the TA (SEQ ID NO: 136) also were used in the same manner as described above. 64 kinds of modified EAD2 obtained by modifying the EAD2, 1024 kinds of modified c-Myc obtained by modifying the c-Myc, and 1232 kinds of modified TA obtained by modifying the TA were provided.
The immobilization was performed by binding the 3′ end of the spacer with an electrode of one microarray and binding the 3′ end of the known polynucleotide or the modified polynucleotide with the 5′ end of the spacer. 100 each of the EAD2, the c-Myc, and the TA as controls of redox activity and 5 each of the modified polynucleotides were randomly immobilized on the microarray chip.
Then, redox reactions were measured three times under the same conditions using the same microarray chip. Specifically, a tris buffer solution (pH7.4) containing 2 mmol/L hydrogen peroxide was added to the microarray chip, and electrical signals generated by the redox reactions were measured as currents.
Results of these are shown in
In graphs of
New DNAzyme having high redox activity was screened using the microarray chip of Example 2.
From the result obtained by the three-time measurements in Example 2, a modified polynucleotide exerting high activity compared with EAD2 was found. This result is shown in
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 Japanese patent application No. 2011-448562, filed on Jul. 4, 2011, the disclosure of which is incorporated herein its entirety by reference.
According to the present invention, the presence or absence or the intensity of redox activity of a nucleic acid molecule to be evaluated can be evaluated easily. Moreover, by such evaluation method according to the present invention, a plurality of nucleic acid molecules can be evaluated at the same time. Thus, an intended nucleic acid molecule can be screened efficiently, for example. As mentioned above, for example, the nucleic acid molecule having redox activity can be used as a substitute for an enzyme such as peroxidase and is thus useful in various fields such as clinical medical care, food, and environment.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011-148562 | Jul 2011 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2012/066912 | 7/2/2012 | WO | 00 | 1/8/2014 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2013/005723 | 1/10/2013 | WO | A |
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| 1553188 | Dec 2004 | CN |
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