JP2010041951A - Method for detecting target molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting a target molecule in high precision using a label without influencing affinity of an aptamer for the target molecule when the target molecule is detected by using a nucleic acid to be specifically bound with the target molecule. <P>SOLUTION: The method for detecting a target molecule by using a nucleic acid to be specifically bound with the target molecule includes (a) a process for forming a complex of the target molecule, a first nucleic acid and a second nucleic acid to which a label is added and (b) a process for detecting the complex formed by the process (a) by measuring a signal sent from the label. The first nucleic acid has an aptamer region to be specifically bound with the target molecule and a binding region with the second nucleic acid and the second nucleic acid has a binding region with the first nucleic acid. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for detecting a target molecule using an aptamer that can specifically bind to the target molecule.

  When detecting biomolecules such as nucleic acids and proteins, detection is performed using the detection molecule that can specifically bind to the target molecule to be detected using the interaction between the detection molecule and the target molecule. The method is generally done. For example, target molecules are detected by various methods such as aggregation methods such as immunoturbidimetry, precipitation methods, ELISA, immunochromatography, etc., using antibodies with target molecules as antigens.

  In recent years, an attempt has been made to detect by using an aptamer capable of specifically binding to a target molecule instead of an antibody as a detection molecule. Since aptamers are functional single-stranded nucleic acids, they can be easily synthesized using an automatic nucleic acid synthesizer or the like, and have the advantage of being more stable than antibodies consisting mainly of proteins. Also, the design is relatively easy.

When the target molecule is detected, the signal emitted from the label is measured in advance by labeling the detection molecule with a fluorescent substance or a radioisotope, and the detection molecule is highly accurate and simple. The target molecule forming a complex with can be detected. Aptamer labeling is usually performed by directly binding a labeling substance such as a fluorescent substance to a nucleic acid, as in the case of conventional PCR primer and probe labeling. Moreover, in order to suppress the influence on the interaction between the aptamer and the target substance due to the binding of the labeling substance, a method of providing an appropriate linker between the aptamer region and the labeling site is also disclosed (for example, non-patent document). See 1 or 2.)
Yoshihiro Miwa (editor), Yodosha, “How to choose and use fluorescent / luminescent reagents”, pages 60-61. Komatsu, Y., 9 others, Bioorganic & Medicinal Chemistry, 2008, Vol. 16, No. 2, pp. 941-949.

  When a labeling substance such as a fluorescent substance is directly labeled on the aptamer, even if a linker is provided between the aptamer region and the labeling site, the aptamer and the target molecule are labeled by the effect of the labeling substance. There is a problem that the bondability of the is easily lowered. In particular, when measurement is performed in a solution system as in the case of using a single molecule fluorescence analysis method, the binding force between the target molecule and the aptamer is significantly reduced, and accurate measurement is often difficult.

  In the present invention, when a target molecule is detected using a nucleic acid that specifically binds to the target molecule, the target molecule can be detected with high accuracy using a label without affecting the binding between the nucleic acid and the target molecule. It is an object to provide a method that can be detected.

  As a result of intensive studies to solve the above problems, the present inventors have found that an aptamer region that specifically binds to a target molecule, a first nucleic acid having a binding region with a second nucleic acid, and a first nucleic acid A complex having an aptamer and a target molecule while suppressing the effect on the binding property between the aptamer and the target molecule by using a second nucleic acid having a binding region of Since the body can be labeled, it has been found that the target molecule can be detected with high accuracy by measuring the signal emitted from the label, and the present invention has been completed.

That is, the present invention
(1) A method for detecting a target molecule using a nucleic acid capable of specifically binding to the target molecule, wherein (a) the target molecule, the first nucleic acid, and the second nucleic acid to which a label is added And (b) detecting the complex formed in the step (a) by measuring a signal emitted from the label. The nucleic acid has an aptamer region that specifically binds to the target molecule and a binding region with the second nucleic acid, and the second nucleic acid has a binding region with the first nucleic acid. A method for detecting a target molecule, characterized by
(2) The method for detecting a target molecule according to (1) above, wherein the binding region of the second nucleic acid with the first nucleic acid contains an artificial nucleotide,
(3) The method for detecting a target molecule according to (1) or (2), wherein the Kd value (equilibrium dissociation constant) between the first nucleic acid and the second nucleic acid is 30 nM or less,
(4) The Kd value (equilibrium dissociation constant) of the target molecule, the first nucleic acid, and the second nucleic acid is 50 nM or less, according to any one of (1) to (3), A method for detecting a target molecule,
(5) The method for detecting a target molecule according to any one of (2) to (4), wherein the artificial nucleotide is Bridged Nucleic Acid (BNA),
(6) Any one of (2) to (5) above, wherein artificial nucleotides and natural nucleotides are alternately arranged in the binding region of the second nucleic acid to the first nucleic acid. A method for detecting the target molecule as described,
(7) The method for detecting a target molecule according to any one of (2) to (5), wherein the binding region of the second nucleic acid with the first nucleic acid is composed only of artificial nucleotides,
(8) One or more selected from the group consisting of a nucleotide having adenine as a base, a nucleotide having thymine as a base, and a nucleotide having uracil as a base in the binding region of the second nucleic acid to the first nucleic acid. The method for detecting a target molecule according to any one of the above (1) to (7), comprising only the nucleotide of
(9) The binding region of the second nucleic acid with the first nucleic acid is a region consisting only of adenine nucleotides, a region consisting only of thymine nucleotides, or a region consisting only of uracil nucleotides. 1) A method for detecting a target molecule according to any one of (7),
(10) One or more selected from the group consisting of a nucleotide having adenine as a base, a nucleotide having thymine as a base, and a nucleotide having uracil as a base in the binding region of the first nucleic acid to the second nucleic acid. The method for detecting a target molecule according to any one of the above (1) to (9), comprising only the nucleotide of
(11) In the first nucleic acid, the binding region with the second nucleic acid is a region consisting of only adenine nucleotides, a region consisting of only thymine nucleotides, or a region consisting of only uracil nucleotides. 1) A method for detecting a target molecule according to any one of (9),
(12) The method for detecting a target molecule according to any one of (1) to (11) above, wherein the binding region of the first nucleic acid to the second nucleic acid is 5 to 30 bases in length. ,
(13) The first nucleic acid has a region that specifically binds to the binding region with the second nucleic acid on the opposite side of the binding region with the second nucleic acid across the aptamer region. The method for detecting a target molecule according to any one of the above (5) to (12),
(14) After the step (a) forms a complex of the first nucleic acid and the second nucleic acid, the target molecule is reacted to cause the target molecule, the first nucleic acid, and the The method for detecting a target molecule according to any one of (1) to (13), which is a step of forming a complex with a second nucleic acid,
(15) After the step (a) forms a complex of the first nucleic acid and the target molecule, the target nucleic acid, the first nucleic acid, and the target nucleic acid are reacted by reacting the second nucleic acid. The method for detecting a target molecule according to any one of (1) to (13), which is a step of forming a complex with a second nucleic acid,
(16) Any of the above (1) to (15), wherein the label is a fluorescent label, and the signal emitted from the label in the step (b) is measured by single molecule fluorescence analysis A method for detecting a target molecule according to
(17) The method for detecting a target molecule according to the above (16), wherein the single molecule fluorescence analysis method is a fluorescence polarization analysis method (FIDA-PO),
(18) A method for detecting a nucleic acid, wherein (a′1) a step of forming a complex of the first nucleic acid and a second nucleic acid to which a label is added, (b′1) the step Detecting the complex formed in (a′1) by measuring a signal emitted from the label, wherein the first nucleic acid comprises an aptamer region, and the second A nucleic acid detection method comprising a binding region with a nucleic acid, and wherein the second nucleic acid has a binding region with the first nucleic acid,
(19) A method for measuring the pharmacokinetics of a nucleic acid administered into a living body, comprising: (a′2-1) a step of administering a first nucleic acid into a living body and collecting a biological sample after a predetermined time; (A′2-2) A second nucleic acid to which a label is added is added to the biological sample collected in the step (a′2-1), and the complex with the first nucleic acid in the biological sample is added. And (b′2) detecting the complex formed in the step (a′2-2) by measuring a signal emitted from the label. The first nucleic acid has an aptamer region and a binding region for the second nucleic acid, and the second nucleic acid has a binding region for the first nucleic acid. A method for measuring the pharmacokinetics of a nucleic acid,
Is provided.

  In the target molecule detection method of the present invention, in order to label a complex composed of an aptamer and a target molecule, it is not necessary to add a label directly to the aptamer. For this reason, by using the method for detecting a target molecule of the present invention, it becomes possible to detect the target molecule with high accuracy while remarkably suppressing the influence on the binding property between the aptamer and the target molecule.

  In the present specification, “target molecule” means a molecule to be detected. The target molecule in the present invention may be a nucleic acid, a protein, or a low molecular compound such as vitamins or hormones. Further, it may be a naturally occurring molecule or an artificially synthesized compound such as a pharmaceutical or environmental hormone.

  In the present specification, “nucleic acid” means a polynucleotide (nucleotide chain) obtained by phosphodiester bonding of two or more nucleotides. The nucleic acid in the present invention may be composed of only natural nucleotides (naturally occurring nucleotides) such as DNA and RNA, and is synthesized from RNA contained in a biological sample or the like using reverse transcriptase. The synthesized cDNA or the like may be used, or it may be a chimeric chain containing both deoxyribonucleotides and ribonucleotides. Further, it may be an artificial nucleic acid partially or entirely containing an artificial nucleotide. In addition, the side chain or the like may be modified with a functional group such as an amino group, or may be labeled with a protein or a low molecular weight compound.

  In the present specification, “artificial nucleotide” means an artificially synthesized nucleotide having a structure different from that of a natural nucleotide but capable of functioning in the same manner as a natural nucleotide. Here, “can function in the same way as natural nucleotides” means that a polymer can be formed by a phosphodiester bond or the like, as in the case of natural nucleotides, and it is composed of part or all of artificial nucleotides. The PCR primer or probe means that it can hybridize with the template nucleic acid in the same manner as the PCR primer or probe constructed using only natural nucleotides.

  Examples of such an artificial nucleotide include Bridged Nucleic Acid (BNA), a nucleotide in which the 4′-position oxygen atom of a natural nucleotide is substituted with a sulfur atom, and a 2′-position hydroxyl group of a natural ribonucleotide in a methoxy group And nucleotides substituted with hexitol Nucleic Acid (HNA), peptide nucleic acid (PNA) and the like.

  In the present specification, for example, the term “nucleotide having adenine as a base” may be either a nucleotide having adenine as a base or a nucleotide derivative. A natural deoxyribonucleotide having adenine as a base, adenine It means a natural ribonucleotide having a base, a derivative of a natural nucleotide having adenine as a base, an artificial nucleotide having adenine as a base, and the like.

  In the present specification, the “aptamer” means any molecule that has a main component as a single-stranded nucleic acid and binds to a target molecule with high affinity and high specificity. In addition, in order to improve functions such as degradation suppression for nucleic acids which are main constituent components, it may be modified with a functional group such as a methyl group, an ethyl group, an aldehyde group, a carbonyl group, or an organic compound. In addition, an inorganic substance or the like may be added.

  Further, in the present specification, “hybrid” means a double strand between the same species and between different species formed between any of the above nucleic acids, and includes DNA-DNA, DNA-RNA, RNA-RNA and the like. included.

  The target molecule detection method of the present invention is a method for detecting a target molecule using a nucleic acid that specifically binds to the target molecule, wherein (a) the target molecule, the first nucleic acid, and a label are added. Forming a complex with the second nucleic acid, and (b) detecting the complex formed in the step (a) by measuring a signal emitted from the label. And the first nucleic acid has an aptamer region that specifically binds to the target molecule, and a binding region between the second nucleic acid, and the second nucleic acid is the first nucleic acid. And a coupling region.

  For example, even when an aptamer binds strongly to a target molecule in an unlabeled state where no label is added, this aptamer is added when labeled with a labeling substance such as a fluorescent substance. Due to the effect of the labeled substance, the binding to the target molecule becomes weak, and accurate measurement may be difficult.

  In contrast, the target molecule detection method of the present invention adds a label to the first nucleic acid that functions as an aptamer and the second nucleic acid that binds to the first nucleic acid, and the target molecule and the first nucleic acid are added. A ternary complex of the (aptamer) and the second nucleic acid (labeled nucleic acid) is formed, and the ternary complex is detected based on the label of the second nucleic acid. Thus, instead of adding a label directly to an aptamer, a label is added to a third nucleic acid molecule that is neither an aptamer nor a target molecule, and a region for binding to the nucleic acid molecule is added to the aptamer. The effect of the added label on the binding between the target molecule and the aptamer can be remarkably suppressed. For this reason, the target molecule and the aptamer (first nucleic acid) and the labeled nucleic acid (second nucleic acid) can form a ternary complex, and the target molecule and the aptamer can bind strongly, and the label By using, it becomes possible to measure and analyze the degree of binding between the aptamer (first nucleic acid) and the target molecule very accurately.

  The reason why such an effect can be obtained by the method for detecting a target molecule of the present invention is not clear, but by adding a labeling substance to the second nucleic acid instead of the first nucleic acid that binds to the target molecule, This is presumably because the target substance does not affect the three-dimensional structure of the aptamer region in the first nucleic acid, and the three-dimensional structure of the aptamer region does not change. Here, “the three-dimensional structure does not change” does not mean that the three-dimensional structure does not change at all, but affects the detection and measurement of the complex composed of the target molecule, the first nucleic acid and the second nucleic acid. This means that the change does not substantially affect and does not substantially change.

  The first nucleic acid in the target molecule detection method of the present invention is a nucleic acid having an aptamer region that specifically binds to the target molecule and a binding region of the second nucleic acid. In the first nucleic acid, the binding region with the second nucleic acid may be on the 5 'side or 3' side of the aptamer region.

  In the present invention, the aptamer region means a polynucleotide region having a base sequence that can function as an aptamer that specifically binds to a target molecule. However, in the present invention, specifically binding means that it can bind specifically to the extent that it can be normally used for detection and purification of the target molecule, and crosses with other substances. Also good.

  The base sequence of the aptamer region may have the same base sequence as a known aptamer, or may be arbitrarily designed so as to have a binding ability with a desired target molecule. The base sequence of a single-stranded nucleic acid having a specific tertiary structure that can bind to a specific target molecule can be designed by a conventional method.

  The base sequence of the binding region with the second nucleic acid in the first nucleic acid is not particularly limited as long as it has a small effect on the binding property between the aptamer region and the target molecule, but has adenine as a base. It preferably consists of only one or more nucleotides selected from the group consisting of nucleotides, nucleotides having thymine as a base, and nucleotides having uracil as a base. This is because it can be expected that the effect on the binding property between the aptamer region and the target molecule can be reduced as compared with nucleotides having guanine, cytosine, or the like as a base. Among these, a region consisting only of adenine nucleotides, a region consisting only of thymine nucleotides, or a region consisting only of uracil nucleotides is more preferable.

  Further, the length of the binding region of the first nucleic acid with the second nucleic acid has a small influence on the binding property between the aptamer region and the target molecule, and can realize sufficient binding property with the second nucleic acid. If it is, it will not specifically limit and it can determine suitably considering the kind etc. of an aptamer area | region. For example, when the length of the binding region with the second nucleic acid is 5 to 30 bases, the binding property to the second nucleic acid is improved while suppressing the influence on the binding property between the aptamer region and the target molecule. can do.

  The second nucleic acid in the target molecule detection method of the present invention is a nucleic acid to which a label is added and which has a binding region with the first nucleic acid. Here, as a label added to the second nucleic acid, it means all labels used for detecting a substance. Specifically, a fluorescent label, a luminescent label, a label using a radioisotope, and the like can be appropriately selected from those generally used for labeling nucleic acids.

The addition of a fluorescent label or a luminescent label can be performed by binding a labeling substance such as a fluorescent substance, a chemiluminescent substance, or an enzyme used for a chemiluminescent reaction to the second nucleic acid. The binding of the labeling substance to the second nucleic acid can be performed by a conventional method. The labeling position may be a site where the binding between the first nucleic acid and the target molecule is not impaired by the labeling substance bound to the second nucleic acid when the first nucleic acid and the second nucleic acid are bound. For example, there is no particular limitation. For example, when the binding region of the first nucleic acid with the second nucleic acid is on the 5 ′ end side, it is preferable to bind the labeling substance to the 5 ′ end side of the second nucleic acid. On the other hand, when the binding region of the first nucleic acid with the second nucleic acid is on the 3 ′ end side, it is preferable to bind the labeling substance to the 3 ′ end side of the second nucleic acid. On the other hand, labeling using a radioisotope can be performed, for example, by synthesizing a second nucleic acid using a nucleotide having a radioisotope such as H ³ , P ³² , or C ¹⁴ .

  The fluorescent substance used for labeling is not particularly limited, and can be appropriately selected from fluorescent substances usually used for labeling of nucleic acids and the like. Examples of such fluorescent substances include FITC (fluorescein isothiocyanate), fluorescein, rhodamine, TAMRA, NBD, TMR (tetramethylrhodamine) and the like. The chemiluminescent substance used for labeling may be a substance that emits light by an inorganic reaction such as luminol, or a luminescent substance that is catalyzed by an enzyme such as luciferin or coelenterazine. Furthermore, the enzyme used for labeling can be appropriately selected from enzymes usually used in detection of a target substance utilizing an enzyme reaction. Examples of such enzymes include alkaline phosphatase, peroxidase, luciferase and the like. The second nucleic acid in the present invention is preferably a nucleic acid to which a fluorescent label is added from the viewpoint of detection sensitivity and safety.

  The base sequence of the binding region of the second nucleic acid with the first nucleic acid is not particularly limited as long as it has a small effect on the binding property between the aptamer region and the target molecule, but has adenine as a base. It preferably consists of only one or more nucleotides selected from the group consisting of nucleotides, nucleotides having thymine as a base, and nucleotides having uracil as a base. This is because it can be expected that the effect on the binding property between the aptamer region and the target molecule can be reduced as compared with nucleotides having guanine, cytosine, or the like as a base. Among these, a region consisting only of adenine nucleotides, a region consisting only of thymine nucleotides, or a region consisting only of uracil nucleotides is more preferable.

  The binding region with the second nucleic acid in the first nucleic acid is a region capable of hybridizing with the second nucleic acid, and the binding region with the first nucleic acid in the second nucleic acid is the first region. A region that can hybridize with a nucleic acid. Specifically, the binding region with the second nucleic acid in the first nucleic acid is a polynucleotide region having a base sequence highly complementary to the binding region with the first nucleic acid in the second nucleic acid.

  The binding property between the first nucleic acid and the second nucleic acid is preferably high. This is because, when the binding property between the first nucleic acid and the second nucleic acid is low, the detection sensitivity of the target molecule bound to the first nucleic acid may be lowered. In addition, when there are many free second nucleic acids labeled in the reaction system, noise increases at the time of measurement in the subsequent step (b), which may impair measurement sensitivity and accuracy. Specifically, if the Kd value (equilibrium dissociation constant) between the first nucleic acid and the second nucleic acid is 30 nM or less, the amount of the first nucleic acid or the second nucleic acid added to the reaction system, the amount of the labeling substance This is because the complex of the target molecule, the first nucleic acid and the second nucleic acid can be sufficiently formed by adjusting the type or the like as appropriate, and this complex can be detected with high accuracy. In the present invention, the Kd value of the first nucleic acid and the second nucleic acid is preferably 25.8 nM or less, more preferably 0.1 to 25.8 nM, and more preferably 0.7 to 25.M. More preferably, it is 8 nM.

  In the present invention, it is also preferable that the binding properties of the target molecule, the first nucleic acid, and the second nucleic acid are high. Specifically, if the Kd value of the target molecule, the first nucleic acid, and the second nucleic acid is 50 nM or less, the target molecule can be detected with very high accuracy, and not only qualitative detection but also quantification More reliable results can be obtained even in automatic or semi-quantitative detection. In the present invention, the Kd values of the target molecule, the first nucleic acid, and the second nucleic acid are preferably 20 nM or less, and more preferably 10 nM or less.

  In order to maintain sufficiently high binding between the first nucleic acid and the second nucleic acid, the binding region of the second nucleic acid with the first nucleic acid preferably contains an artificial nucleotide. This is because by including an artificial nucleotide, the binding property to the first nucleic acid can be made higher than that of a region consisting only of natural nucleotides. In the present invention, it is particularly preferable that the binding region with the first nucleic acid in the second nucleic acid contains BNA. BNA is a nucleic acid in which part of a natural nucleotide is cross-linked, and is a cross-linked artificial nucleotide in which the 2′-position oxygen atom and the 4′-position carbon atom of the ribose ring are bonded via methylene. Includes a certain locked nucleic acid (LNA). Since BNA has such a crosslinked structure, the ability of the polynucleotide to bind to DNA or RNA can be dramatically improved by including BNA in the polynucleotide.

  In the present invention, as long as the Kd value of the first nucleic acid and the second nucleic acid can be 30 nM or less, the number and sequence of artificial nucleotides included in the binding region of the second nucleic acid with the first nucleic acid are It is not particularly limited. For example, natural nucleotides and artificial nucleotides may be randomly arranged, may be alternately arranged, or may be arranged so as to have a part consisting only of natural nucleotides and a part consisting only of artificial nucleotides. . All the nucleotides constituting the region may be artificial nucleotides. In the present invention, it is preferable that natural nucleotides and artificial nucleotides are alternately arranged in the binding region of the second nucleic acid with the first nucleic acid. This is because the binding to the first nucleic acid can be enhanced while suppressing the influence due to the difference in the three-dimensional structure between the artificial nucleotide and the natural nucleotide.

  In the present invention, as the binding region of the second nucleic acid to the first nucleic acid, for example, a region consisting only of a natural nucleotide having adenine as a base and BNA having adenine as a base, or a thymine as a base. It is preferably any one of a region consisting only of natural nucleotides and BNA having thymine as a base, and a region consisting only of natural nucleotides having uracil as a base and BNA having uracil as a base. In addition, a region where BNA and natural nucleotides are alternately arranged is more preferable.

  Further, when the binding region of the second nucleic acid with the first nucleic acid contains BNA, the first nucleic acid is located on the opposite side of the binding region with the second nucleic acid across the aptamer region. It is also preferable to have a region that specifically binds to the binding region with the second nucleic acid. By having regions consisting of base sequences complementary to each other across the aptamer region, these regions constitute a double strand, and the first nucleic acid can take a more stable intramolecular loop structure, This is because the stability of the whole molecule can be further improved. Since an artificial nucleic acid having BNA has a very high binding ability to DNA or RNA, it can enter between these double strands and bind to a binding region with a second nucleic acid.

  In the formation of the complex of the target molecule, the first nucleic acid and the second nucleic acid in the step (a), first, the first nucleic acid and the second nucleic acid are combined to form a complex, and then the target molecule is formed. May be formed by reacting the first nucleic acid and the target molecule to form a complex, and then formed by reacting the second nucleic acid. Since the second nucleic acid used in the present invention has little influence on the binding property between the aptamer region of the first nucleic acid and the target molecule, the first nucleic acid and the second nucleic acid are bound first. This is because the complex of the target molecule, the first nucleic acid and the second nucleic acid can be satisfactorily formed in any case where the first nucleic acid and the target nucleic acid are bound first. .

  In the present invention, the first nucleic acid and the target nucleic acid can be reacted with each other after the first nucleic acid and the target nucleic acid are bound to form a complex. The reaction with the second nucleic acid is performed immediately before the measurement operation, even if the reaction requires a long period of time or when there is a waiting time after the reaction between the first nucleic acid and the target nucleic acid. As a result, it is possible to avoid a problem caused by the deterioration of the sign and perform highly accurate detection. For example, in the case of detecting a target molecule in a living body, first, after the first nucleic acid is administered into the living body, it waits for a certain period of time, and the first nucleic acid administered with the target molecule in the living body Are combined. Collecting a biological sample after a predetermined time, and reacting a second nucleic acid to the biological sample to label and detect a target molecule bound to the first nucleic acid contained in the biological sample. Can do. In addition, since the first nucleic acid is not labeled, when administered to a living body, for example, in the blood or an organ, it is possible to maintain a higher order structure without being affected by the label. Will be able to show the proper pharmacokinetics. Thus, by using the target molecule detection method of the present invention, whether or not the target molecule and the nucleic acid specifically binding to the target molecule (the first nucleic acid in the present invention) are bound is determined in It can be confirmed not only in vitro but also in vivo.

  Thus, before detecting the target molecule in the living body using the first nucleic acid, it is preferable to examine the pharmacokinetics of the first nucleic acid in advance. As described above, the pharmacokinetics of the first nucleic acid is that after the first nucleic acid is administered into the living body, the biological sample is collected after a predetermined period of time, and the second nucleic acid is reacted with the biological sample. Thus, a complex of the first nucleic acid and the second nucleic acid contained in the biological sample is formed. By detecting this complex, the first nucleic acid in the biological sample can be detected, and thus the pharmacokinetics of the first nucleic acid can be measured.

  The formed complex of the target molecule, the first nucleic acid and the second nucleic acid can be detected by measuring a signal emitted from the label. In the present specification, “signal” means a signal that can be appropriately detected and measured by appropriate means, and includes fluorescence, radioactivity, chemiluminescence, and the like. For example, when the second nucleic acid is labeled with a fluorescent substance, the formed complex is irradiated with excitation light having a wavelength suitable for the fluorescent property of the fluorescent substance, and the second nucleic acid has The complex can be detected by measuring the fluorescence emitted from the fluorescent substance. Measurement of each signal emitted from a fluorescent substance, a chemiluminescent substance, or a labeling substance such as an enzyme used for a chemiluminescent reaction can be performed by a conventional method using a known measuring means.

  In the present invention, the target nucleic acid and the first nucleic acid formed by labeling the second nucleic acid with a fluorescent substance and measuring the fluorescence emitted from the fluorescent substance using single molecule fluorescence analysis. And the second nucleic acid complex can be easily detected. In the present specification, “single-molecule fluorescence analysis” refers to fluorescence cross-correlation method FCS (Fluorescence correlation spectroscopy), fluorescence intensity distribution analysis method FIDA (Fluorescence Intensity Distribution Analysis), and fluorescence polarization analysis method FIDA-Penity POF. (Distribution Analysis-polarization). Measurement and analysis by single-molecule fluorescence analysis eliminates the need for solid-phase or separate labeling of the nucleic acid that binds to the target molecule, enabling measurement and analysis in an environment closer to the living body become.

  FCS is an analysis method for obtaining a translational diffusion time of a fluorescent molecule from a detected fluorescent signal. Since the translational diffusion time varies depending on the size of the molecule, it is mainly used to monitor changes in size such as molecular interaction, decomposition, and aggregation. For example, since the second nucleic acid forming the complex has a larger molecular size than the free nucleic acid not forming the complex, the ratio of the complex can be determined by calculating the translational diffusion time of each molecule. And the quantity, that is, the ratio and quantity of the target molecule can be obtained.

  FIDA is an analysis method for obtaining the fluorescence intensity per molecule of fluorescent molecules. When two or more types of fluorescent molecules having different fluorescence intensities exist in the reaction system, the number and ratio of the molecules having the respective fluorescence intensities. Can also be calculated. For this reason, for example, when the fluorescence intensity of the fluorescent substance labeled with the second nucleic acid is changed by forming a complex, the complex (that is, the target molecule) is obtained by obtaining the fluorescence intensity of each molecule. The ratio and quantity can be obtained.

  FIDA-PO is an analysis method in which FIDA and fluorescence polarization analysis are combined, and the degree of fluorescence polarization and the number of molecules of fluorescent molecules can be determined. For example, the second nucleic acid that forms a complex has a larger molecule than the free nucleic acid that does not form a complex, and the rotational movement is slow, so the degree of fluorescence polarization increases. Therefore, by obtaining the fluorescence polarization degree of each molecule, the ratio and quantity of the complex, that is, the ratio and quantity of the target molecule can be obtained.

  Thus, in the single molecule fluorescence analysis method, the reaction is performed by utilizing the fact that the size and fluorescence intensity per molecule differ between the second nucleic acid forming the complex and the free second nucleic acid. By determining the ratio and quantity of the complex in the system, the target molecule forming the complex can be detected. In addition, when a label is directly added to an aptamer that binds to a target molecule as in the conventional method, the size of the target molecule is smaller than the size of the labeled aptamer, and the free labeled aptamer and the target molecule When the size difference between the labeled aptamer and the bound aptamer is small, FCS and FIDA-PO cannot distinguish the complex from the free labeled aptamer, and the target molecule cannot be detected accurately. There wasn't. On the other hand, the target molecule detection method of the present invention has a size per molecule of a free second nucleic acid and a complex composed of the target molecule, the first nucleic acid and the second nucleic acid. Is larger than, for example, the difference in size per molecule between the labeled free first nucleic acid and the complex composed of the labeled first nucleic acid and the target molecule. By using FIDA-PO, it is possible to obtain a measurement result with higher reliability than the conventional method. In particular, in the present invention, it is preferable to measure and analyze with FIDA-PO.

  In single-molecule fluorescence analysis methods such as FCS, FIDA, and FIDA-PO, a free second nucleic acid, a complex composed of a first nucleic acid and a second nucleic acid, a target molecule, a first nucleic acid, and a second nucleic acid are used. It is possible to detect and distinguish the complex composed of the nucleic acid of each. For this reason, in the present invention, detection of the complex in the step (b) is performed by using a single molecule fluorescence analysis method, so that detection with higher accuracy is possible. In particular, whether the complex that emits the signal by analyzing the measured signal by two-component analysis or the like is a complex composed of the first nucleic acid and the second nucleic acid (binary complex) It is also possible to distinguish whether it is a complex (triple complex) composed of a target molecule, a first nucleic acid and a second nucleic acid. For example, when the ratio of the three-component complex to the sum of the two-component complex and the three-component complex is not less than a predetermined value (for example, 10%), the target molecule, the first nucleic acid, and the second It can be determined that the target molecule is present in the sample. Thus, the method for detecting a target molecule of the present invention uses a single molecule fluorescence analysis method to detect a complex consisting of a target molecule, a first nucleic acid, and a second nucleic acid semi-quantitatively. Therefore, it is also possible to determine the Kd values of the target molecule and the first nucleic acid.

  In addition, in single-molecule fluorescence analysis methods such as FCS, FIDA, and FIDA-PO, two-component analysis and the like are used to distinguish between a free second nucleic acid and a second nucleic acid forming a complex. You can also Here, the “second nucleic acid forming a complex” means all the second nucleic acids other than free ones, and a complex composed of the first nucleic acid and the second nucleic acid. And a complex composed of the target molecule, the first nucleic acid, and the second nucleic acid. That is, after a second nucleic acid is added to a sample and reacted, the sample is analyzed by single molecule fluorescence analysis to form a complex with the free second nucleic acid in the sample. Therefore, the first nucleic acid in the sample can be detected. Specifically, for example, after the first nucleic acid is administered into the living body, the biological sample is collected after a predetermined period of time, and the second nucleic acid is reacted with the biological sample. By performing a two-component analysis of this biological sample by single molecule fluorescence analysis, a complex of the first nucleic acid and the second nucleic acid contained in the biological sample can be detected. By detecting this complex, the first nucleic acid in the biological sample can be detected, and thus the pharmacokinetics of the first nucleic acid can be measured.

  For example, the target molecule, the first nucleic acid, the second nucleic acid, and the like can be obtained by labeling the target molecule in advance using a fluorescent substance having a fluorescence characteristic different from that of the fluorescent substance used for labeling the second nucleic acid. It is also possible to detect and quantify the complex consisting of using fluorescence cross-correlation spectroscopy FCCS (Fluorescence cross correlation spectroscopy).

EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
The measurement using FIDA-PO was performed using MF20 (manufactured by OLYMPUS) with PBS containing 0.05% Tween 20 as a measurement buffer and a measurement volume of 30 μL. The measurement conditions were 10 times for 10 seconds and the laser power was 100W.

[Example 1]
The TAT protein was detected by the target molecule detection method of the present invention using RNA having an aptamer region that specifically binds to the TAT protein.
First, RNA with 12 adenines added to the 5 ′ end of the aptamer region that specifically binds to the TAT protein (T-aptamer) and a TAMRA label at the 5 ′ end of the aptamer region that specifically binds to the TAT protein RNA (5TAM-aptamer) was synthesized. Furthermore, RNA (BNA probe) in which TAMRA label was added to the 5 ′ end of RNA (BNA1) made by alternately binding BNA with thymine as a base and natural ribonucleotide with thymine as a base was synthesized. That is, the TAT protein is a target molecule, the T-aptamer corresponds to the first nucleic acid, and the BNA probe corresponds to the second nucleic acid. The 5TAM-aptamer is obtained by adding a label directly to a conventional aptamer. 1 schematically shows the structure of T-aptamer, FIG. 2 shows the structure of 5TAM-aptamer, and FIG. 3 shows the structure of BNA probe. In the figure, an asterisk indicates TAMRA. “T” represents BNA (thymine type BNA) having thymine as a base, and “t” represents thymine.

T-aptamer 5 nM and TAT protein 0-50 nM were respectively added to BNA probe 5 nM, reacted at room temperature for 15 minutes, and measured with FIDA-PO. Similarly, 5 TAM-aptamer 8 nM and TAT protein 0 to 50 nM were added, reacted at room temperature for 15 minutes, and measured with FIDA-PO.
The measurement results are shown in Table 1. FIG. 4 shows the result of reacting the BNA probe, T-aptamer and TAT protein, FIG. 5 shows the result of reacting the BNA probe and TAT protein, and the result of reacting 5TAM-aptamer and TAT protein. Are shown in FIG.
From Table 1 and FIG. 4, it was confirmed that the fluorescence polarization degree increased as the TAT protein concentration increased. This indicates that the molecular weight of the fluorescent molecule in the reaction solution increases depending on the TAT protein concentration, that is, a molecule (complex) in which the T-aptamer and the TAT protein are bound depending on the TAT protein concentration. Indicates an increase.
Further, from Table 1 and FIG. 5, it was confirmed that the degree of fluorescence polarization did not change much even when the concentration of TAT protein was increased. This indicates that the BNA probe and the TAT protein are not directly bound. That is, the binding between the T-aptamer and the TAT protein in FIG. 4 is specific. Furthermore, when the TAT protein concentration was 50 nM or less, almost no change was observed in the degree of fluorescence polarization, suggesting that the concentration of 50 nM or less was hardly affected by viscosity.
Furthermore, from Table 1 and FIG. 6, although a tendency of increasing the degree of fluorescence polarization depending on the TAT protein concentration was observed, the degree of fluorescence polarization was much higher than when the BNA probe, T-aptamer and TAT protein were reacted. The rate of increase decreased. For example, when the TAT protein concentration is 50 nM, the amount of increase in the degree of fluorescence polarization compared with the case of 0 nM was 129.8 in FIG. 4, but only 32.4 in FIG. 6. This indicates that when the aptamer is directly fluorescently labeled, the binding force between the aptamer and the target molecule is weakened.

Thereafter, the reaction between the BNA probe, the T-aptamer, and the TAT protein is inhibited by adding an unlabeled aptamer (only an aptamer region that specifically binds to the TAT protein and does not have an adenine addition sequence). The assay was performed. Specifically, 500 nM or 1000 nM of unlabeled aptamer was added to 5 nM of BNA probe, 5 nM of T-aptamer and 25 nM of TAT protein, respectively, and a competitive reaction was performed at room temperature for 15 minutes, and measurement was similarly performed with FIDA-PO. .
The measurement results are shown in Table 2 and FIG. As a result, the fluorescence polarization degree was 202.2 and 203.3 for unlabeled aptamers 500 nM and 1000 nM, respectively, whereas that for unlabeled aptamer 0 nM was 273.0. This indicates that the formation of the ternary complex was inhibited by the unlabeled aptamer. This confirmed that the ternary complex was specifically bound to the TAT protein.

[Example 2]
The influence of the binding properties of the first nucleic acid and the second nucleic acid on the detection of the target molecule was examined.
First, two types of probes (BNA7mer probe and BNA3mer probe) in which a TAMRA label is added to the 5 ′ end of RNA having the base sequence shown in Table 3, and the 5 ′ end of DNA having the base sequence shown in Table 3 are used. A TAMRA label was added to synthesize a probe (DNA probe).
The T-aptamer used in Example 1 was added to 1 nM of each of these three types of probes at different concentrations in the range of 5 to 1000 nM and reacted at room temperature for 15 minutes. In the same manner as in Example 1, FIDA- Measurement was performed at PO, and each Kd value was calculated. The calculated results are shown in Table 3. Of the three types of probes, the BNA7mer probe had the strongest binding force with the T-aptamer, and then the BNA3mer probe had the strongest binding force, and the DNA probe containing no artificial nucleotide had the lowest binding force.

Next, using these probes having different Kd values, an experiment was conducted to confirm the binding state of the probe, T-aptamer, and TAT protein. Tn aptamer 1 nM and TAT protein 0 to 50 nM were added to 1 nM each of BNA7mer probe, BNA3mer probe, and DNA probe, respectively, and reacted at room temperature for 15 minutes. Was measured.
Table 4 shows the measurement results. FIG. 8 shows the result of reacting BNA7mer probe, T-aptamer and TAT protein, FIG. 9 shows the result of reacting BNA3mer probe, T-aptamer and TAT protein, and FIG. 9 shows the result of reacting BNA3mer probe, T-aptamer and TAT protein. The results of reacting with the protein are shown in FIG.
As shown in FIGS. 8 and 9, when the BNA7mer probe or the BNA3mer probe was used, the binding of the probe, T-aptamer, and TAT protein was confirmed depending on the TAT protein concentration. With the BNA7mer probe, the degree of fluorescence polarization increased by 175.5 (when the TAT protein concentration was 50 nM) (FIG. 8). On the other hand, the BNA3mer probe increased by 81.0 at the maximum (when the TAT protein concentration was 50 nM) (FIG. 9). However, although it was confirmed that the BNA3mer probe was bound depending on the TAT protein concentration, the binding force was weaker than that of the BNA7mer probe. On the other hand, in the DNA probe, significant binding with T-aptamer and TAT protein was not confirmed. (FIG. 10).
From these data, the stronger the binding force between the T-aptamer and the fluorescently labeled probe such as BNA or DNA, that is, the smaller the Kd value, the more the triplet complex of the fluorescently labeled probe, TAT protein and T-aptamer. It was shown that the degree of binding of the body increased. When the binding force between the fluorescently labeled probe and the T-aptamer is weak, the fluorescently labeled probe is present alone in the solution without binding to the T-aptamer, and mixed with the ternary complex. . For this reason, when the binding force is small, the proportion of molecules having a small molecular weight is larger than when the binding force is large. When measurement is performed with FIDA-PO, the average of these values is obtained, and thus the degree of fluorescence polarization is small when the binding force is weak.

[Example 3]
In addition to the probes shown in Table 3, probes having a ratio of thymine-type BNA and thymine were designed, and the probes shown in Table 5 were synthesized. The Kd value of the binding of these seven kinds of probes to the T-aptamer was measured in the same manner as in Example 2. Further, the Kd value of the triple binding of the probe, T-aptamer, and TAT protein was also measured in the same manner. Table 5 shows the measurement results.

Of these, as in Example 2, 1 nM of T-aptamer and 0 to 50 nM of TAT protein were added to 1 nM of each of the BNA2mer probe, BNA4mer probe, BNA5mer probe, and BNA15mer probe, and reacted at room temperature for 15 minutes. And measurement was performed with FIDA-PO.
As a result, the BNA2mer probe finally confirmed significant binding when the TAT protein concentration was 50 nM. On the other hand, when the BNA3mer probe, the BNA4mer probe, the BNA5mer probe, and the BNA15mer probe were used, it became clear that the binding force was stronger than that of the BNA2mer probe. In particular, the BNA4mer probe, the BNA5mer probe, and the BNA15mer probe were confirmed to have sufficiently good binding although they were slightly weaker than the BNA7mer probe.
From these results, in the method for detecting a target molecule of the present invention, the binding property between the first nucleic acid and the second nucleic acid is important, particularly in the binding between the first nucleic acid and the second nucleic acid. If the Kd value is about 30 nM or less, the target molecule can be detected well. In particular, if the binding between the target molecule, the first nucleic acid and the second nucleic acid is about 50 nM or less, the target molecule can be detected with higher accuracy. It is clear that it can be detected.

[Example 4]
In FIDA-PO, a complex composed of a first nucleic acid and a second nucleic acid (bipartite complex), and a complex composed of a target molecule, the first nucleic acid and the second nucleic acid (triple complex) ) And the target molecule was detected.
Specifically, T-aptamer 5 nM and TAT protein 0 to 1000 nM were added to BNA15mer probe 5 nM, respectively, and allowed to react at room temperature for 15 minutes in the same manner as in Example 1, followed by measurement with FIDA-PO. It was. The signal data obtained as a result of the measurement is subjected to two-component analysis, and the sum of the complex composed of the first nucleic acid and the second nucleic acid and the complex composed of the target molecule, the first nucleic acid and the second nucleic acid. The ratio of each complex to was calculated.
The results are shown in Table 6. In the table, “Frac.K1 [%]” indicates the ratio of the presence of the BNA15mer probe and T-aptamer complex, and “Frac.K2 [%]” indicates the BNA15mer probe. The ratio at which a ternary complex of T-aptamer and TAT protein exists is shown.
As is clear from these results, the target molecule detection method of the present invention uses a single-molecule fluorescence analysis method such as FIDA-PO to semi-quantitatively target molecule, the first nucleic acid, and the first nucleic acid. It is possible to distinguish and detect a complex composed of two nucleic acids and a complex composed of a first nucleic acid and a second nucleic acid.

[Example 5]
The first nucleic acid is RNA (AT-aptamer) with 15 adenines added to the 5 ′ end of the aptamer region that specifically binds to the TAT protein and 15 thymines added to the 3 ′ end. RNA with a TAMRA label added to the 3 ′ end of 15-mer RNA (BNA1) obtained by alternately binding thymine-based BNA and thymine-based natural ribonucleotides (3′-end TAMRA-labeled BNA probe) ), TAT protein was detected using the target molecule detection method of the present invention. Furthermore, the binding of TAT protein with RNA (3TAM-aptamer) added with TAMRA labeling at the 3 ′ end of the aptamer region specifically binding to TAT protein was also confirmed. FIG. 11 is a diagram schematically showing the structure of AT-aptamer, and FIG. 12 is a diagram schematically showing the structure of 3TAM-aptamer. In the figure, an asterisk indicates TAMRA. Since the adenine 15mer at the 5 ′ end and the thymine 15mer at the 3 ′ end of the AT-aptamer are complementary sequences, they are theoretically bound in a complementary manner to form a double strand.

AT-aptamer 5 nM and TAT protein 0-50 nM were added to 3′-terminal TAMRA-labeled BNA probe 5 nM, respectively, and allowed to react at room temperature for 15 minutes. In the same manner as in Example 1, measurement was performed with FIDA-PO. went. Similarly, 3 TAM-aptamer 8 nM and TAT protein 0 to 50 nM were added, reacted at room temperature for 15 minutes, and measured with FIDA-PO.
Table 7 shows the measurement results. FIG. 13 shows the result of reacting the 3′-end TAMRA-labeled BNA probe, AT-aptamer and TAT protein, FIG. 14 shows the result of reacting the 3′-end TAMRA-labeled BNA probe and TAT protein, and 5TAM- The results of the reaction between the aptamer and the TAT protein are shown in FIG.
From Table 7 and FIG. 13, it was confirmed that a ternary complex in which the 3 ′ end TAMRA-labeled BNA probe, the AT-aptamer, and the TAT protein were bound was formed depending on the TAT protein concentration.
Further, from Table 7 and FIG. 14, it was revealed that the 3 ′ terminal TAMRA-labeled BNA probe and the TAT protein do not bind directly and do not cause non-specific binding. At the same time, it became clear that TAT protein had no viscosity effect at this concentration.
Furthermore, from Table 7 and FIG. 15, it was clarified that the binding between 3TAM-aptamer and TAT protein was observed, but the binding was very weak. For example, when the TAT protein concentration is 50 nM, the amount of increase in the degree of fluorescence polarization compared to the case of 0 nM is 47.1 in FIG. 13 but only 9.8 in FIG. This indicates that when the aptamer is directly fluorescently labeled, the binding force between the aptamer and the target molecule is weakened.

[Example 6]
In the formation of a complex of the target molecule, the first nucleic acid, and the second nucleic acid, the first nucleic acid and the second nucleic acid are bound first, and the first nucleic acid and the target nucleic acid are bound first. The case where it was made to compare was compared.
First, sample 5A was prepared by adding 5 nM of T-aptamer to BNA probe 5 nM and reacting for 15 minutes at room temperature, and then adding 50 nM TAT protein and reacting for 15 minutes at room temperature.
On the other hand, TAT aptamer 5 nM was added to TAT protein 50 nM and allowed to react at room temperature for 15 minutes, and then BNA probe 5 nM was further added and reacted at room temperature for 15 minutes to obtain sample 5B.
As a control, sample 5C was prepared by adding 5 nM of T-aptamer to 5 nM of BNA probe and allowed to react for 15 minutes at room temperature. Sample 5D was prepared by adding 50 nM of TAT protein to 5 nM of BNA probe and reacted for 15 minutes at room temperature. Sample 5E was prepared by adding only 5 nM BNA probe and reacting at room temperature for 15 minutes.
Each sample was measured by FIDA-PO in the same manner as in Example 1. Table 8 shows the measurement results. Thus, the case where the TAT protein is bound after the BNA probe and the T-aptamer are bound first than the case where the BNA probe is bound after the TAT protein and the T-aptamer are bound first. Although it was slightly better, it became clear that both of the methods could form a complex of BNA probe, T-aptamer and TAT protein.

[Example 7]
Mice were administered T-aptamer (FIG. 1) or AT-aptamer (FIG. 11), and the change in concentration in the living body was examined.
First, a calibration curve for examining each aptamer concentration was prepared. Specifically, each concentration of aptamer and BNA probe 5 nM was added and allowed to react at room temperature for 15 minutes, and measurement was performed with FIDA-PO. Table 9 shows the measurement results.

  Here, it was assumed that the hybridization reaction between the BNA probe and the aptamer was the following equilibrium reaction.

  Fitting was performed on the assumption that the following equation was established with the Kd value, bottom value, and top value as constants. In the formula, y represents the degree of fluorescence polarization, x represents the aptamer concentration [nM] added, and A0 represents the initial concentration of BNA probe (2 nM).

  The fitting results are shown in FIG. In FIG. 16, A (nM) on the horizontal axis indicates aptamer concentration, and B on the vertical axis indicates the degree of fluorescence polarization. Each point represents a measured value, and the curve represents a calibration curve obtained by fitting. For the convenience of creating a logarithmic graph, a point with an aptamer concentration of 0 nM is expressed as 0.01 nM. From the calibration curve, it was predicted that Kd = 5.2 ± 1.5 nM.

Mice were administered 1 mg / mL of T-aptamer or AT-aptamer for each individual, blood was collected every 0, 1, 8, 24, 48, and 72 hours, and the aptamer concentration in the blood was measured. The collected blood was diluted 50-fold with PBS containing 0.05% Tween 20, added 5 nM of TAMRA-labeled BNA7mer probe, reacted at room temperature for 15 minutes, and measured with FIDA-PO. The measurement results are shown in FIG.
Furthermore, concentration of the obtained results was calculated based on the above calibration curve, and the concentration of aptamer in blood was calculated. The estimated blood concentrations are shown in Table 10.
As described above, since the first nucleic acid (aptamer) that binds to the target molecule used in the present invention is not labeled, it is possible to measure the more accurate pharmacokinetics without the influence of the label. .

  By using the target molecule detection method of the present invention, it is possible to detect the target molecule with high accuracy while significantly suppressing the influence on the binding property between the aptamer and the target molecule. It can be used in fields such as biochemistry, molecular biology, and clinical testing that detect and analyze biomolecules.

It is the figure which showed the structure of T-aptamer typically. It is the figure which showed typically the structure of 5TAM-aptamer. It is the figure which showed the structure of the BNA probe typically. In Example 1, it is the figure which showed the result of having made the BNA probe, T-aptamer, and TAT protein react. In Example 1, it is the figure which showed the result of having made the BNA probe and TAT protein react. In Example 1, it is the figure which showed the result of having made 5TAM-aptamer react with TAT protein. In Example 1, it is the figure which showed the result of the inhibition assay which made unlabeled aptamer react with the reaction of a BNA probe, T-aptamer, and TAT protein. In Example 2, it is the figure which showed the result of having made BNA7mer probe, T-aptamer, and TAT protein react. In Example 2, it is the figure which showed the result of having made BNA3mer probe, T-aptamer, and TAT protein react. In Example 2, it is the figure which showed the result of having made the DNA probe, T-aptamer, and TAT protein react. It is the figure which showed the structure of AT-aptamer typically. It is the figure which showed typically the structure of 3TAM-aptamer. In Example 5, it is the figure which showed the result of having made 3 'terminal TAMRA labeled BNA probe, AT-aptamer, and TAT protein react. In Example 5, it is the figure which showed the result of having made 3 'terminal TAMRA labeled BNA probe and TAT protein react. In Example 5, it is the figure which showed the result of having made 5TAM-aptamer react with TAT protein. It is the figure which showed the calibration curve of the aptamer density | concentration created in Example 7. FIG. In Example 7, it is the figure which showed the measurement result of each aptamer density | concentration in the 50 times dilution liquid of blood.

Claims (19)

A method for detecting a target molecule using a nucleic acid capable of specifically binding to the target molecule,
(A) forming a complex of the target molecule, the first nucleic acid, and the second nucleic acid to which a label is added;
(B) detecting the complex formed in the step (a) by measuring a signal emitted from the label;
Have
The first nucleic acid has an aptamer region that specifically binds to the target molecule, and a binding region of the second nucleic acid;
The method for detecting a target molecule, wherein the second nucleic acid has a binding region with the first nucleic acid.   The method for detecting a target molecule according to claim 1, wherein the binding region of the second nucleic acid with the first nucleic acid contains an artificial nucleotide.   The method for detecting a target molecule according to claim 1 or 2, wherein a Kd value (equilibrium dissociation constant) between the first nucleic acid and the second nucleic acid is 30 nM or less.   The method for detecting a target molecule according to any one of claims 1 to 3, wherein a Kd value (equilibrium dissociation constant) of the target molecule, the first nucleic acid, and the second nucleic acid is 50 nM or less.   The method for detecting a target molecule according to any one of claims 2 to 4, wherein the artificial nucleotide is Bridged Nucleic Acid (BNA).   The target molecule detection according to any one of claims 2 to 5, wherein in the binding region of the second nucleic acid to the first nucleic acid, artificial nucleotides and natural nucleotides are alternately arranged. Method.   The method for detecting a target molecule according to any one of claims 2 to 6, wherein a binding region of the second nucleic acid with the first nucleic acid is composed of only artificial nucleotides.   The binding region of the second nucleic acid to the first nucleic acid is only one or more nucleotides selected from the group consisting of nucleotides having adenine as a base, nucleotides having thymine as a base, and nucleotides having uracil as a base. The method for detecting a target molecule according to claim 1, comprising:   The binding region of the second nucleic acid to the first nucleic acid is a region consisting only of adenine nucleotides, a region consisting only of thymine nucleotides, or a region consisting only of uracil nucleotides. The method for detecting a target molecule according to any one of the above.   The binding region of the first nucleic acid with the second nucleic acid is only one or more nucleotides selected from the group consisting of nucleotides having adenine as a base, nucleotides having thymine as a base, and nucleotides having uracil as a base. The method for detecting a target molecule according to any one of claims 1 to 9, wherein   10. The binding region of the first nucleic acid with the second nucleic acid is a region consisting only of adenine nucleotides, a region consisting only of thymine nucleotides, or a region consisting only of uracil nucleotides. The method for detecting a target molecule according to any one of the above.   The method for detecting a target molecule according to any one of claims 1 to 11, wherein a binding region with the second nucleic acid in the first nucleic acid has a length of 5 to 30 bases.   The first nucleic acid has a region that specifically binds to the binding region with the second nucleic acid on the opposite side of the binding region with the second nucleic acid across the aptamer region. The method for detecting a target molecule according to any one of claims 5 to 12.   After the step (a) forms a complex of the first nucleic acid and the second nucleic acid, the target molecule, the first nucleic acid, and the second nucleic acid are reacted by reacting the target molecule. The method for detecting a target molecule according to any one of claims 1 to 13, which is a step of forming a complex with a nucleic acid.   After the step (a) forms a complex of the first nucleic acid and the target molecule, the target nucleic acid, the first nucleic acid, and the second nucleic acid are reacted by reacting the second nucleic acid. The method for detecting a target molecule according to any one of claims 1 to 13, which is a step of forming a complex with a nucleic acid.   The target label according to any one of claims 1 to 15, wherein the label is a fluorescent label, and the signal emitted from the label in the step (b) is measured by single molecule fluorescence analysis. Detection method.   The method for detecting a target molecule according to claim 16, wherein the single-molecule fluorescence analysis is Fluorescence Intensity Distribution Analysis-polarization (FIDA-PO). A method for detecting a nucleic acid comprising:
(A′1) forming a complex of the first nucleic acid and the second nucleic acid to which a label is added;
(B′1) detecting the complex formed in the step (a′1) by measuring a signal emitted from the label;
Have
The first nucleic acid has an aptamer region and a binding region of the second nucleic acid;
The method for detecting a nucleic acid, wherein the second nucleic acid has a binding region with the first nucleic acid. A method for measuring the pharmacokinetics of a nucleic acid administered in vivo,
(A′2-1) administering a first nucleic acid to a living body and collecting a biological sample after elapse of a predetermined time;
(A′2-2) A second nucleic acid to which a label is added is added to the biological sample collected in the step (a′2-1), and the complex with the first nucleic acid in the biological sample is added. Forming a body;
(B′2) detecting the complex formed in the step (a′2-2) by measuring a signal emitted from the label;
Have
The first nucleic acid has an aptamer region and a binding region of the second nucleic acid;
The method for measuring pharmacokinetics of a nucleic acid, wherein the second nucleic acid has a binding region with the first nucleic acid.

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