Method for Detecting Oligonucleotides Using Probes

Abstract

Provided is an oligonucleotide measurement method that is simpler and has higher sensitivity and superior specificity and quantitative properties compared with conventional measurement methods. Also provided is an oligonucleotide measurement method having superior specificity which makes it possible to distinguish an intact target oligonucleotide (unmodified form) from a metabolite thereof so as to detect only the unmodified form.　In a hybridization method using a capture probe and an assist probe, a capture probe and an assist probe each having a short base length within a certain range, and in particular, an assist probe having a short base length within a certain range that is not generally considered, are used, and a defective nucleotide site in a metabolite of a nucleic acid drug and the assist probe are hybridized in a specific positional relationship. As a result, a target oligonucleotide in a sample can be detected, and the target oligonucleotide and a metabolite of the nucleic acid drug can be distinguished from each other.

Description

Technical Field

[0001] The present invention relates to a method for detecting or quantifying oligonucleotides with high sensitivity, excellent specificity and quantification.

Background Art

[0002] Nucleic acid drugs have attracted great attention in recent years as new therapeutic agents for various diseases that have been difficult to treat until now, such as gene diseases and intractable diseases, because they cause sequence-specific gene silencing (Non-Patent Document 1, Ando 2012).

[0003] In pharmacokinetics / pharmacodynamics (PK / PD) screening tests in the exploratory stage of drug development, in safety tests, pharmacological tests and pharmacokinetic tests in the non-clinical stage, and in the clinical stage, the drug concentration in animal or human biological samples administered with the drug is measured. Also, when obtaining approval as a new pharmaceutical, it is necessary to acquire data on the drug concentration in biological samples and submit materials on safety and pharmacokinetics in accordance with the guidelines stipulated by the Ministry of Health, Labour and Welfare Ordinance.

[0004] Ando et al. reported that by labeling nucleic acid drugs with positron-emitting radionuclides, the behavior in vivo can be three-dimensionally analyzed non-invasively and in real time (Non-Patent Document 1, Ando 2012). Also, Healey et al. reported that they achieved a lower limit of quantification of 0.01 pg / μL using the stem-loop RT-PCR method (Non-Patent Document 3, Chen 2005) (Non-Patent Document 2, Healey 2014).

[0005] However, in recent years, the development of artificial nucleic acids and delivery materials has enabled treatment at low doses. As a result, the drug concentration in biological samples has become lower than before, and in order to detect these low-concentration drugs, a more sensitive measurement system has been required. Furthermore, in order to comply with the guidelines stipulated by the Ministry of Health, Labour and Welfare Ordinance, the measurement system needs to have high quantification that is independent of the operator. Therefore, measurement by the PCR method, which is a semi-quantitative method, has been unsuitable for the purpose of detecting these low-concentration drugs.

[0006] In addition, in the conventional measurement system, when the oligonucleotide used in nucleic acid medicine is metabolized and degraded from the 5' or 3' end, it cannot be distinguished from the oligonucleotide to be measured (that is, the intact oligonucleotide that has not been degraded from the 5' or 3' end by metabolism (hereinafter sometimes simply referred to as the "intact" oligonucleotide)), and there has been a problem that the accurate drug concentration cannot be measured.

[0007] To distinguish between intact oligonucleotides to be measured and their metabolites and specifically measure nucleic acid pharmaceuticals with biological activity, Yu et al. developed a hybridization-ligation ELISA method (Non-Patent Document 4, Yu 2002). In this method, a "template" oligonucleotide containing a sequence complementary to the oligonucleotide to be measured and a "ligation probe" are used. The "template" oligonucleotide has an additional 9-mer nucleotide adjacent to the nucleotide at the 5'-end of the complementary sequence and biotin at the 3'-end. The "ligation probe" is a 9-mer oligonucleotide having a sequence complementary to the additional 9-mer nucleotide, with a phosphate at the 5'-end and digoxigenin at the 3'-end. Therefore, when the oligonucleotide to be measured is intact, the intact oligonucleotide and the ligation probe hybridize without a gap on the template oligonucleotide. By treating the product of this hybridization with ligase, the intact oligonucleotide and the ligation probe are ligated. On the other hand, when the oligonucleotide to be measured is metabolized and nucleotides on the 3'-end side are missing, this ligation does not occur. The above ligation product is bound to a solid phase using biotin, unreacted ligation probes are washed away, and the digoxigenin at the 3'-end of the immobilized ligation product is detected by ELISA (see Figure 1 of Non-Patent Document 4, Yu 2002). Wei et al. disclosed performing S1 nuclease treatment after ligase treatment to improve the specificity of the hybridization-ligation ELISA method (Non-Patent Document 5, Wei 2006). However, the hybridization-ligation ELISA method requires designing the optimal sequence of the ligation probe in consideration of the sequence of the oligonucleotide to be measured, so it was cumbersome and unsuitable for multiplexing.

[0008] To solve the above problems of the hybridization-ligation ELISA method, Omori et al. developed a method of decomposing and removing products derived from metabolites of target oligonucleotides using S1 nuclease or the like and measuring products derived from the remaining intact target oligonucleotides (Patent Document 1). In this method, the target oligonucleotide to be measured is hybridized with a complementary nucleic acid probe (3'-complementary sequence of the target sequence-5'), or an arbitrary base such as polyA (first polynucleotide) is added to the target oligonucleotide to be measured and hybridized with a complementary nucleic acid probe (3'-complementary sequence of the target oligonucleotide + complementary sequence of the first polynucleotide-5'), and single-strand specific nucleases such as S1 nuclease are used to decompose and remove incomplete hybridization products, and the nucleic acid probes contained in the remaining complete hybridization products are measured.

[0009] The method of Omori et al. is a method for measuring oligonucleotides that is more sensitive, specific, and quantitative than conventional methods such as the hybridization-ligation ELISA method. However, it is complicated in that single-strand specific nucleases such as S1 nuclease are used, and it is disadvantageous in that it takes additional incubation time. Furthermore, in the "Guidance on the Conduct of Non-Clinical Safety Tests for the Clinical Trials and Applications for Approval of Manufacturing and Sale of Pharmaceuticals" by the Ministry of Health, Labour and Welfare, when the cross-reactivity of metabolites exceeds 10%, a toxicity test is required. As described above, the ability to measure the cross-reactivity of metabolites at 10% or less is an important criterion in pharmaceutical development. However, in conventional hybridization-based methods such as the hybridization-ligation ELISA method, the cross-reactivity of metabolites is still high, and it has been difficult to suppress and detect the cross-reactivity to stably reach 10% or less.

[0010] In order to develop a method for detecting or quantifying oligonucleotides that meets the above high requirements and has high versatility in pharmaceutical development, the inventors have developed a method for detecting or quantifying oligonucleotides that is simple, highly sensitive, quantitative, and excellent in metabolite discrimination ability, and completed the present invention.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

[0013] In conventional signal detection methods (ligand binding assays such as hybridization method and ligation method, qPCR), it is difficult to distinguish between oligonucleotides with a partial deletion in the sequence of the target oligonucleotide to be measured, particularly oligonucleotides with a deletion at the 5'- or 3'-end (such as metabolites), and intact target oligonucleotides (unmodified forms). There was a problem that both metabolites and unmodified forms were measured. In addition, the methods of Yu et al. and Omori et al. that solved this problem also required enzymatic treatment of samples and had problems in terms of simplicity. On the other hand, liquid chromatography-mass spectrometry (LC-MS) and HPLC-UV methods can distinguish and measure metabolites and intact target oligonucleotides, but they have the drawback of insufficient sensitivity. The problem to be solved by the present invention is to provide a method for measuring oligonucleotides that is simpler, more sensitive, and superior in specificity and quantitativeness compared to conventional measurement methods. Another problem to be solved by the present invention is to provide a method for measuring oligonucleotides that is excellent in specificity and can distinguish between the above-mentioned unmodified form and metabolite and detect only the unmodified form. Means for Solving the Problems

[0014] As a method for measuring anti-drug antibodies, a double antigen bridging immunoassay method using a capture drug antibody and a tracer drug antibody is known (Patent Document 3). In this method, a capture drug antibody and a tracer drug antibody specifically bind to an analyte (anti-drug antibody) contained in a sample, thereby forming a trimer of capture drug antibody - analyte - tracer drug antibody. After binding the capture drug antibody to a solid phase and removing the free tracer drug antibody, a signal proportional to the amount of analyte in the sample can be obtained by detecting the signal derived from the label contained in the tracer drug antibody. The present inventors examined a hybridization method (see Patent Document 4) using a capture probe and an assist probe as a method for measuring a target oligonucleotide, and thus a nucleic acid drug, in a sample (hereinafter, may be referred to as the CP-AP method for convenience). In the CP-AP method, a first nucleic acid probe contained in the capture probe and a second nucleic acid probe contained in the assist probe specifically hybridize to a nucleic acid drug (target oligonucleotide) contained in the sample, thereby forming a trimer of capture probe - nucleic acid drug - assist probe. Generally, as in the case of the primer chain length of the PCR method, also in the probe chain length in a signal detection method based on hybridization, considering the hybridization efficiency between the probe and the target oligonucleotide, the probe chain length is almost always designed to be 15 mer or more. Actually, in the examples of Patent Document 4, the target analyte is a 120 mer oligonucleotide (SEQ ID NO: 3) having a sequence derived from ApoE, the capture probe is a 61 mer oligonucleotide (SEQ ID NO: 4), and the assist probe is an oligonucleotide consisting of a 21 mer oligonucleotide and a 59 mer tag sequence (SEQ ID NO: 5). Further, in Patent Document 4, the distinction between the target analyte and its metabolite is not recognized as an issue, and as a result, naturally, the positional relationship between the missing site of the metabolite compared with the target analyte and the probe has not been examined. The inventors of the present invention have intensively studied to realize the measurement of oligonucleotides and thus nucleic acid drugs by the CP-AP method, and have used a capture probe and an assist probe with a short base length within a certain range, particularly an assist probe with a short base length within a certain range that is not usually considered. Furthermore, by hybridizing the nucleotide deletion site in the metabolite of the nucleic acid drug and the assist probe in a specific positional relationship, it has been found that not only can the nucleic acid drug (target oligonucleotide) in the sample be detected, but surprisingly, it is also possible to distinguish it from the metabolite of the nucleic acid drug. The inventors of the present invention have further found that by using the present invention, the cross-reactivity of the metabolite of the nucleic acid drug can be suppressed to an extremely low level.

[0015] From the above, the present invention has the following configuration. [Embodiment 1] A method for measuring a target oligonucleotide in a sample by combining a capture probe and an assist probe based on the principle of hybridization, and a method for distinguishing and measuring a target oligonucleotide that retains its full-length sequence and its metabolite, The capture probe includes a solid phase and a first nucleic acid probe immobilized on the solid phase, The assist probe includes a tag or label and a second nucleic acid probe linked to the tag or label, Among the nucleotides of the second nucleic acid probe, the nucleotide closest to the tag or label forms a base pair with the nucleotide at the 3'-end or 5'-end of the target oligonucleotide, In the metabolite, one or more consecutive nucleotides including the nucleotide at the 3'-end or 5'-end are missing, The second nucleic acid probe can hybridize to a portion containing the nucleotide that is missing in the metabolite of the target oligonucleotide, The first nucleic acid probe can hybridize to a portion other than the above-mentioned portion of the target oligonucleotide, The capture probe, the target oligonucleotide, and the assist probe form a complex, Method. [Embodiment 2] When measuring the target oligonucleotide in a sample by distinguishing it from a metabolite lacking one or more nucleotides from its 3'-end, the method according to Embodiment 1, wherein the second nucleic acid probe included in the assist probe is linked to a tag or a label via the nucleotide at the 5'-end. [Embodiment 3] When measuring the target oligonucleotide in a sample by distinguishing it from a metabolite lacking one or more nucleotides from its 5'-end, the method according to Embodiment 1, wherein the second nucleic acid probe included in the assist probe is linked to a tag or a label via the nucleotide at the 3'-end. [Embodiment 4] A method for detecting a target oligonucleotide in a sample by distinguishing it from a metabolite lacking one or more nucleotides from its 3'-end or 5'-end, comprising the following steps: (i) Contacting a sample containing a target oligonucleotide or a metabolite lacking one or more nucleotides from its 3'-end or 5'-end with a capture probe for capturing the target oligonucleotide and an assist probe for detecting the target oligonucleotide to form a complex of the capture probe, the target oligonucleotide, and the assist probe; wherein the capture probe includes a solid phase and a first nucleic acid probe immobilized on the solid phase; the assist probe includes a tag or a label and a second nucleic acid probe linked to the tag or the label; the sequence of the second nucleic acid probe is complementary to a partial sequence of the target oligonucleotide, and the partial sequence includes nucleotides that are lacking in the metabolite; the sequence of the first nucleic acid probe is complementary to a sequence other than the partial sequence of the target oligonucleotide; and the tag or the label is linked to the nucleotide at the end of the second nucleic acid probe, and the nucleotide at the end forms a base pair with the nucleotide at the end of the target oligonucleotide that is lacking in the metabolite when the target oligonucleotide hybridizes with the second nucleic acid probe; and (ii) Detecting the target oligonucleotide in the sample by detecting the complex. [Embodiment 5] When distinguishing and detecting the target oligonucleotide in the sample from metabolites lacking one or more nucleotides from its 3'-end, the second nucleic acid probe is linked to a tag or label via the nucleotide at its 5'-end, and the sequence of the second nucleic acid probe is complementary to the sequence containing the 3'-end of the target oligonucleotide, the method according to Embodiment 4. [Embodiment 6] When distinguishing and detecting the target oligonucleotide in the sample from metabolites lacking one or more nucleotides from its 5'-end, the second nucleic acid probe is linked to a tag or label via the nucleotide at its 3'-end, and the sequence of the second nucleic acid probe is complementary to the sequence containing the 5'-end of the target oligonucleotide, the method according to Embodiment 4. [Embodiment 7] A method for detecting a target oligonucleotide in a sample, comprising the following steps: (i) Contacting the sample with a capture probe for capturing the target oligonucleotide and an assist probe for detecting the target oligonucleotide to form a complex of the capture probe, the target oligonucleotide, and the assist probe; wherein the capture probe comprises a solid phase and a first nucleic acid probe immobilized on the solid phase; the assist probe comprises a tag or label and a second nucleic acid probe linked to the tag or label; the sequence of the second nucleic acid probe is complementary to a partial sequence of the target oligonucleotide containing the nucleotide at the end of the target oligonucleotide; the sequence of the first nucleic acid probe is complementary to a sequence other than the partial sequence of the target oligonucleotide; and The tag or label is linked to the nucleotide at the end of the second nucleic acid probe, and the nucleotide at the end of the second nucleic acid probe forms a base pair with the nucleotide at the end of the target oligonucleotide when the target oligonucleotide hybridizes with the second nucleic acid probe; and (ii) detecting the target oligonucleotide in the sample by detecting the complex. [Embodiment 8] The sequence of the second nucleic acid probe is complementary to the 3'-side partial sequence of the target oligonucleotide including the nucleotide at the 3'-end of the target oligonucleotide, the sequence of the first nucleic acid probe is complementary to the sequence other than the 3'-side partial sequence of the target oligonucleotide, and the tag or label is linked to the nucleotide at the 5'-end of the second nucleic acid probe, the method according to Embodiment 7. [Embodiment 9] The sequence of the second nucleic acid probe is complementary to the 5'-side partial sequence of the target oligonucleotide including the nucleotide at the 5'-end of the target oligonucleotide, the sequence of the first nucleic acid probe is complementary to the sequence other than the 5'-side partial sequence of the target oligonucleotide, and the tag or label is linked to the nucleotide at the 3'-end of the second nucleic acid probe, the method according to Embodiment 7 or 8. [Embodiment 10] The second nucleic acid probe included in the assist probe is 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, or 10 bases in length, the method according to any one of Embodiments 1 to 9. [Embodiment 11] The first nucleic acid probe included in the capture probe is 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases, 21 bases, 22 bases, 23 bases, 24 bases or 25 bases in length, the method according to any one of Embodiments 1 to 10. [Embodiment 12] The capture probe includes an adapter or spacer between the first nucleic acid probe and the solid phase, the method according to any one of Embodiments 1 to 11. [Embodiment 13] The assist probe includes a tag having a base sequence complementary to part or all of one of a pair of self - assemblable signal - amplifying probes, The method according to any one of Embodiments 1 to 12, further comprising the following steps: (i) Adding a pair of self - assemblable signal - amplifying probes having complementary base sequence regions that can hybridize to each other to the complex, and forming a probe polymer bound to the tag of the assist probe contained in the complex; and (ii) Detecting the probe polymer. [Embodiment 14] The method according to Embodiment 13, wherein at least one of the pair of self - assemblable signal - amplifying probes contains a poly - T sequence. [Embodiment 15] The method according to Embodiment 13 or 14, wherein at least one of the pair of self - assemblable signal - amplifying probes is labeled with a labeling substance. [Embodiment 16] The pair of self - assemblable signal - amplifying probes consists of a first signal - amplifying probe and a second signal - amplifying probe, The first signal - amplifying probe contains three or more nucleic acid regions, and in order from the 5'-terminal side, contains at least nucleic acid region X, nucleic acid region Y, and nucleic acid region Z or a nucleic acid region Z containing a poly - T sequence, The second signal - amplifying probe contains three or more nucleic acid regions, and in order from the 5'-terminal side, contains at least nucleic acid region X', which is complementary to nucleic acid region X, nucleic acid region Y', which is complementary to nucleic acid region Y, and nucleic acid region Z', which is complementary to nucleic acid region Z or a nucleic acid region Z' containing a poly - A sequence, The method according to any one of Embodiments 13 to 15. [Embodiment 17] A detection kit for detecting a target oligonucleotide, comprising a capture probe, an assist probe, and a pair of signal amplification probes having complementary base sequence regions capable of hybridizing with each other and capable of forming a probe polymer by self-assembly, wherein the capture probe includes a solid phase and a first nucleic acid probe immobilized on the solid phase, the assist probe includes a tag having a base sequence complementary to a part or all of one of the pair of signal amplification probes, and a second nucleic acid probe linked to the tag, the sequence of the second nucleic acid probe is complementary to a partial sequence of the target oligonucleotide including the nucleotides at the ends of the target oligonucleotide, the sequence of the first nucleic acid probe is complementary to a sequence other than the partial sequence of the target oligonucleotide, and the tag is linked to the nucleotide at the end of the second nucleic acid probe, and the nucleotide at the end of the second nucleic acid probe forms a base pair with the nucleotide at the end of the target oligonucleotide when the target oligonucleotide hybridizes with the second nucleic acid probe. Detection kit. [Embodiment 18] The detection kit according to Embodiment 17, wherein the first nucleic acid probe includes an adapter or a spacer between the first nucleic acid probe and the solid phase. [Embodiment 19] A detection kit for measuring a target oligonucleotide in a sample while distinguishing it from a metabolite lacking one or more nucleotides from its 3'-end, wherein the second nucleic acid probe included in the assist probe is linked to the tag via the nucleotide at its 5'-end. The detection kit according to Embodiment 17 or 18. [Embodiment 20] A detection kit for distinguishing and measuring a target oligonucleotide in a sample from a metabolite lacking one or more nucleotides from its 5'-end, wherein the second nucleic acid probe included in the assist probe is linked to a tag via the nucleotide at its 3'-end, the detection kit according to embodiment 17 or 18. [Embodiment 21] The detection kit according to any one of embodiments 17 to 20, wherein at least one of the pair of signal amplification probes is labeled with a labeling substance. [Embodiment 22] The pair of signal amplification probes consists of a first signal amplification probe and a second signal amplification probe, The first signal amplification probe is a nucleic acid probe containing at least a nucleic acid region X, a nucleic acid region Y, and a nucleic acid region Z containing a poly T sequence or a nucleic acid region Z in this order from the 5'-end side, The second signal amplification probe is a nucleic acid probe containing at least a nucleic acid region X' complementary to the nucleic acid region X, a nucleic acid region Y' complementary to the nucleic acid region Y, and a nucleic acid region Z' complementary to the nucleic acid region Z containing a poly A sequence or a nucleic acid region Z' in this order from the 5'-end side, characterized in that The detection kit according to any one of embodiments 17 to 21. [Embodiment 23] The detection of the target oligonucleotide in the sample is to distinguish and detect the target oligonucleotide in the sample from a metabolite lacking one or more nucleotides from its 3'-end or 5'-end, The sample is a sample containing a target oligonucleotide or a metabolite lacking one or more nucleotides from its 3'-end or 5'-end, The partial sequence contains nucleotides that are missing in the metabolite, and The nucleotide at the end of the second nucleic acid probe to which the tag or label is linked forms a base pair with the nucleotide at the end of the target oligonucleotide that is missing in the metabolite when the target oligonucleotide and the second nucleic acid probe hybridize. The method according to embodiment 7. [Embodiment 24] When detecting a target oligonucleotide in a sample by distinguishing it from a metabolite lacking one or more nucleotides from its 3'-end, the second nucleic acid probe is linked to a tag or label via the nucleotide at its 5'-end, and the sequence of the second nucleic acid probe is complementary to the sequence including the 3'-end of the target oligonucleotide. The method according to Embodiment 23. [Embodiment 25] When detecting a target oligonucleotide in a sample by distinguishing it from a metabolite lacking one or more nucleotides from its 5'-end, the second nucleic acid probe is linked to a tag or label via the nucleotide at its 3'-end, and the sequence of the second nucleic acid probe is complementary to the sequence including the 5'-end of the target oligonucleotide. The method according to Embodiment 23. [Advantages of the Invention]

[0016] According to the present invention, without using an enzyme or the like, detection or quantification of an oligonucleotide can be performed simply, with high sensitivity, and with excellent specificity and quantification in only the step of hybridization between probes. In addition, it is possible to provide a method for detecting or quantifying an oligonucleotide that can distinguish between an oligonucleotide metabolite in which the 5'-side or 3'-side of the target oligonucleotide to be measured is deleted and the unchanged form, has almost no cross-reactivity of the metabolite, and has excellent specificity and quantification for detecting only the unchanged form. [Brief Description of the Drawings]

[0017]

Figure 1

[0018] (Sample) The "sample" used in the method of the present invention is a body fluid such as whole blood, serum, plasma, lymph, urine, saliva, tear fluid, sweat, gastric juice, pancreatic juice, bile, pleural effusion, synovial fluid, cerebrospinal fluid, marrow fluid, or a tissue such as liver, kidney, lung, heart, etc. of human, monkey, dog, pig, rat, guinea pig, or mouse. Preferably, the sample is whole blood, serum, plasma, or urine of human, monkey, dog, pig, rat, guinea pig, or mouse, preferably human. More preferably, the sample is whole blood, serum, plasma, or urine of human, monkey, dog, pig, rat, guinea pig, or mouse that has been administered a medicament containing the target oligonucleotide, preferably human.

[0019] (Target oligonucleotide) In the present specification, the term "target oligonucleotide" means an intact oligonucleotide (intact target oligonucleotide / unchanged form) to be measured. That is, the term "target oligonucleotide" does not include metabolites to be distinguished from this. In the present specification, the term "target oligonucleotide" may be either DNA or RNA, either single-stranded or double-stranded, and may be chemically modified as long as it can form a specific hybrid with a nucleic acid probe. Examples of chemical modifications include phosphorothioate modification (S modification), 2'-F modification, 2'-O-Methyl (2'-OMe) modification, 2'-O-Methoxyethyl (2'-MOE) modification, morpholino modification, LNA modification, BNACOC modification, BNANC modification, ENA modification, cEt BNA modification, etc. When the above target oligonucleotide is double-stranded, it is made single-stranded and used in the present invention. The base length of the target oligonucleotide is not limited, but is preferably 12mer, 13mer, 14mer, 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer, 23mer, 24mer, 25mer, 26mer, 27mer, 28mer, 29mer, or 30mer.

[0020] (Capture probe) The "capture probe" used in the present invention is a probe for capturing a target oligonucleotide, and includes a nucleic acid probe and a solid phase adjacent to the nucleotide at the 3'-end or 5'-end of the nucleic acid probe.

[0021] (Assist probe) The "assist probe" used in the present invention is a probe for detecting a target oligonucleotide, and includes a nucleic acid probe and a tag or label adjacent to the nucleotide at the 5'-end or 3'-end of the nucleic acid probe.

[0022] (Nucleic acid probes included in capture probes and assist probes - regarding the nucleotides constituting them) The nucleic acid probes included in the capture probe and the assist probe are composed of deoxyribonucleotides or ribonucleotides. However, in one aspect of the present invention, each independently contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 locked nucleic acids (LNA) (Figure 1). For example, when the nucleic acid probe has a base length of 5mer, the nucleic acid probe preferably contains 0, 1, 2, 3, 4, or 5 locked nucleic acids (LNA). When the nucleic acid probe has a base length of 6mer to 11mer, the nucleic acid probe preferably contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 locked nucleic acids (LNA).

[0023] (Nucleic acid probe - regarding base length) In one aspect, the nucleic acid probe included in the assist probe has a base length of 4mer, 5mer, 6mer, 7mer, 8mer, 9mer, or 10mer. In another aspect, the nucleic acid probe included in the assist probe has a base length of 5mer, 6mer, 7mer, 8mer, 9mer, or 10mer. The nucleic acid probes included in the capture probes have a base length of 5mer, 6mer, 7mer, 8mer, 9mer, 10mer, 11mer, 12mer, 13mer, 14mer, 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer, 23mer, 24mer, 25mer, or 26mer in one aspect. The nucleic acid probes included in the capture probes have a base length of 5mer, 6mer, 7mer, 8mer, 9mer, 10mer, 11mer, 12mer, 13mer, 14mer, 15mer, or 16mer in another aspect. The nucleic acid probes included in the capture probes have a base length of 5mer, 6mer, 7mer, 8mer, 9mer, or 10mer in yet another aspect.

[0024] (Contact) As used herein, the term "contact" or "contacting step" means placing substances in proximity to each other such that chemical bonds, such as covalent bonds, ionic bonds, metallic bonds, non-covalent bonds, etc., can be formed between one substance and another. In one aspect of the present invention, "contacting" one substance with another means mixing a solution containing one substance with a solution containing the other substance. In the present invention, a complex is formed by contacting a capture probe, a target oligonucleotide, and an assist probe. In one aspect, the step of contacting a sample with a capture probe and an assist probe is performed by incubating a mixture containing the sample, the capture probe, and the assist probe at a temperature that is +2°C to -10°C, +1°C to -9°C, 0°C to -8°C, -1°C to -7°C, -2°C to -6°C, or -3°C to -5°C, or +10°C, +9°C, +8°C, +7°C, +6°C, +5°C, +4°C, +3°C, +2°C, +1°C, 0°C, -1°C, -2°C, -3°C, -4°C, -5°C, -6°C, -7°C, -8°C, -9°C, or -10°C compared to the melting temperature (Tm) of the target oligonucleotide and the nucleic acid probe contained in the capture probe for a certain period of time. For example, when the Tm is 50°C, +2°C to -10°C compared to the Tm means 52°C to 40°C. The incubation time is, in one aspect, 10 seconds to 4 minutes, 20 seconds to 3 minutes, or 30 seconds to 2 minutes, or 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 110 seconds, 120 seconds, 130 seconds, 140 seconds, 150 seconds, 160 seconds, 170 seconds, or 180 seconds.

[0025] (Capture) In the present invention, when a capture probe "captures" a target oligonucleotide, it primarily means that the nucleic acid probe contained in the capture probe hybridizes with the target oligonucleotide. In one aspect, when a capture probe "captures" a target oligonucleotide, it means that the target oligonucleotide is indirectly bound to the solid phase contained in the capture probe or to the solid phase to which an adapter or spacer binds via the nucleic acid probe contained in the capture probe. In the present invention, by directly capturing a target oligonucleotide with a capture probe and further indirectly capturing an assist probe via the target oligonucleotide, a signal proportional to the amount of the target oligonucleotide in a sample can be obtained from the assist probe.

[0026] (Hybridization / Hybridize) As used herein, hybridization of a nucleic acid probe contained in a capture probe or an assist probe to a target oligonucleotide means that a single-stranded nucleic acid probe having a sequence complementary to a part of the sequence binds to a single-stranded target oligonucleotide having a specific base sequence through base pairing to form a double-stranded nucleic acid molecule.

[0027] (Complex) As used herein, when forming a "complex" of a capture probe, a target oligonucleotide, and an assist probe, it means forming a trimer in which a nucleic acid probe contained in the capture probe specifically hybridizes with a part of the target oligonucleotide and a nucleic acid probe contained in the assist probe specifically hybridizes with another part of the target oligonucleotide. Here, specific hybridization of a nucleic acid probe with a part of a target oligonucleotide means that, excluding tags, all bases contained in the nucleic acid probe form pairs with the bases of the target oligonucleotide. In one aspect, all bases contained in the target oligonucleotide form pairs with the bases of the nucleic acid probe contained in the capture probe or the bases of the nucleic acid probe contained in the assist probe.

[0028] (Removal) When detecting a complex of a capture probe, a target oligonucleotide, and an assist probe, if a signal derived from the free assist probe hinders the detection, it is preferable to remove the free assist probe. For example, the free assist probe can be removed by washing the solid phase contained in the capture probe or the solid phase bound via an adapter or a spacer contained in the capture probe. To wash the solid phase, the liquid phase of the reaction solution in which the solid phase is suspended may be separated by centrifuging or filtering the reaction solution. Also, when the solid phase has magnetism, the solid phase can be recovered using a magnet. The washing of the solid phase may be performed a plurality of times as necessary.

[0029] (Detection) To "detect" a target oligonucleotide, a tag or a label contained in the assist probe or a label bound via a tag can be used. Also, when the solid phase contained in the capture probe can emit a signal such as fluorescence, the signal can also be used. The signal from the label or the solid phase may be any signal as long as it is a physically or chemically detectable signal, but an optically detectable signal is preferable in order to achieve high throughput.

[0030] (Self-assembly: PALSAR method) A state in which a plurality of first signal amplification probes form a probe polymer by hybridization with a second signal amplification probe, and a state in which a plurality of second signal amplification probes form a probe polymer by hybridization with a first signal amplification probe are meant.

[0031] (A pair of self-assembling signal amplification probes) The "self - assemblable" pair of signal amplification probes used in the method of the present invention refers to oligonucleotides in which the first signal amplification probe and the second signal amplification probe have complementary base sequence regions capable of hybridizing with each other and can form probe polymers by a self - assembly reaction. Here, "capable of hybridizing" means, in one aspect, being completely complementary in the complementary base sequence region.

[0032] The self - assemblable pair of probes can also be labeled in advance with a labeling substance for detection. Preferably, at least one of the first or second signal amplification probes is labeled with a labeling substance. Examples of such labeling substances include radioisotopes, biotin, digoxigenin, fluorescent substances, luminescent substances, or dyes. Specifically, 125 I and 32 radioisotopes such as P, luminescent and chromogenic substances such as digoxigenin and acridinium ester, luminescent substances such as dioxetane, alkaline phosphatase for using fluorescent substances such as 4 - methylumbelliferyl phosphate, biotin for using fluorescent, luminescent, and chromogenic substances bound to avidin, etc. can be mentioned. Also, it is possible to add a donor fluorescent dye and an acceptor fluorescent dye for using fluorescence resonance energy transfer (FRET) to detect the target oligonucleotide. In one aspect, the labeling substance is biotin, and the labeling of the oligonucleotide is carried out by biotinylating the 5' - end or 3' - end. When the labeling substance is biotin, the substance that specifically binds to the labeling substance is streptavidin or avidin. In one aspect, the labeling substance is not biotin, and the substance that specifically binds to the labeling substance is not streptavidin or avidin.

[0033] In some cases, a complex containing a hybridization product of a target oligonucleotide, a capture probe, and an assist probe in the present invention is contacted with a pair of self-assembling probes consisting of a first and a second signal amplification probe, and the complex is bound to a probe polymer consisting of the first and second signal amplification probes for detection.

[0034] In one aspect, the assist probe used above contains a tag that can bind to one of a pair of self-assembling probes consisting of a first and a second signal amplification probe, and has a role of assisting the binding of the target oligonucleotide to the probe polymer. The first aspect of the assist probe is a probe containing a tag consisting of a sequence complementary to at least the entire sequence or a partial sequence of at least one of the first or second oligonucleotides and a sequence complementary to a partial sequence of the target oligonucleotide.

[0035] (Solid phase) In this specification, examples of the term "solid phase" include substrates such as insoluble fine particles, microbeads, fluorescent fine particles, magnetic particles, microplates, microarrays, slide glasses, and electrically conductive substrates. In one aspect of the present invention, the "solid phase" is fluorescent fine particles, in another aspect it is fluorescent beads, and in still another aspect it is beads having a fluorescent substance on the surface. The "beads having a fluorescent substance on the surface" used in the present invention are not particularly limited as long as they are beads having a fluorescent substance. For example, MicroPlex TM Microspheres of Luminex can be preferably used. It is possible to use one type of bead or multiple types of beads. By using a plurality of types of color-coded beads, the quantification method of the oligonucleotide of the present invention can also be easily multiplexed. In one aspect of the present invention, the "solid phase" is a microplate. Examples of the material of the microplate used in the present invention include, but are not limited to, polystyrene, polypropylene, polycarbonate, and cyclic olefin copolymer. In one aspect of the present invention, the microplate is a coated plate such as a biotin-coated plate, a protein A, G, A / G, and / or L-coated plate, an anti-GST antibody-coated plate, a glutathione, nickel, and / or copper-coated plate, an amine and / or sulfhydryl bond plate, a carboxylated plate, a streptavidin-coated plate, etc. In one aspect, the solid phase is not insoluble fine particles, not microbeads, not fluorescent fine particles, not magnetic particles, not a microplate, not a microarray, not a slide glass, or not a substrate such as an electrically conductive substrate.

[0036] (Adapter) Examples of the "adapter" used in the present invention include, for example, biotin, streptavidin or avidin, and combinations thereof, antigens, antibodies, and combinations thereof, preferably biotin, streptavidin or avidin, and combinations thereof, etc. In one aspect, the adapter is not a nucleic acid such as an oligonucleotide or nucleotide, not biotin, streptavidin or avidin, and combinations thereof, antigens, antibodies, and combinations thereof, not a compound having an amino group or a carboxyl group such as Spacer 9, Spacer 12, Spacer18, Spacer C3, etc. In another aspect, the adapter does not contain a nucleic acid such as an oligonucleotide or nucleotide. Further, in one aspect, streptavidin or avidin is directly immobilized on the solid phase. In another aspect, streptavidin or avidin is not directly immobilized on the solid phase. For example, in the another aspect, streptavidin or avidin is immobilized on the solid phase via a (second) spacer.

[0037] (Spacer) Examples of the "spacer" used in the present invention include nucleic acids such as oligonucleotides and nucleotides, compounds having an amino group or a carboxyl group such as spacers such as Spacer 9, Spacer 12, Spacer18, and Spacer C3, and preferably 5'-Amino-Modifier C12 (12-(4-Monomethoxytritylamino)dodecyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite). For example, a nucleic acid probe having a carboxyl group on the bead surface and an amino group compound added thereto binds to the carboxyl group on the bead surface via the amino group, and the compound having an amino group serves as an example of a spacer. In one aspect, the spacer is not a nucleic acid such as an oligonucleotide or a nucleotide, not biotin, and not a compound having an amino group or a carboxyl group such as spacers such as Spacer 9, Spacer 12, Spacer18, and Spacer C3. In another aspect, the spacer does not contain nucleic acids such as oligonucleotides and nucleotides. Further, in one aspect, the (first) spacer is directly immobilized on the solid phase. In another aspect, the (first) spacer is not directly immobilized on the solid phase. For example, in the other aspect, the (first) spacer is immobilized on the solid phase via biotin, streptavidin or avidin, and combinations thereof. When the spacer is an oligonucleotide, the base length of the oligonucleotide is 4 mer or more and 130 mer or less, 5 mer or more and 90 mer or less, 7 mer or more and 50 mer or less, 10 mer or more and 40 mer or less, 15 mer or more and 30 mer or less, or 4 mer, 5 mer, 6 mer, 7 mer, 8 mer, 9 mer, 10 mer, 11 mer, 12 mer, 13 mer, 14 mer, 15 mer, 16 mer, 17 mer, 18 mer, 19 mer, 20 mer, 21 mer, 22 mer, 23 mer, 24 mer, 25 mer, 26 mer, 27 mer, 28 mer, 29 mer, 30 mer, 31 mer, 32 mer, 33 mer, 34 mer, 35 mer, 36 mer, 37 mer, 38 mer, 39 mer, 40 mer, 41 mer, 42 mer, 43 mer, 44 mer, 45 mer, 46 mer, 47 mer, 48 mer, 49 mer, 50 mer, 51 mer, 52 mer, 53 mer, 54 mer, 55 mer, 56 mer, 57 mer, 58 mer, 59 mer, 60 mer, 61 mer, 62 mer, 63 mer, 64 mer, 65 mer, 66 mer, 67 mer, 68 mer, 69 mer, 70 mer, 71 mer, 72 mer, 73 mer, 74 mer, 75 mer, 76 mer, 77 mer, 78 mer, 79 mer, 80 mer, 81 mer, 82 mer, 83 mer, 84 mer, 85 mer, 86 mer, 87 mer, 88 mer, 89 mer, 90 mer, 91 mer, 92 mer, 93 mer, 94 mer, 95 mer, 96 mer, 97 mer, 98 mer, 99 mer, 100 mer, 101 mer, 102 mer, 103 mer, 104 mer, 105 mer, 106 mer, 107 mer, 108 mer, 109 mer, 110 mer, 111 mer, 112 mer, 113 mer, 114 mer, 115 mer, 116 mer, 117 mer, 118 mer, 119 mer, 120 mer, 121 mer, 122 mer, 123 mer, 124 mer, 125 mer, 126 mer, 127 mer, 128 mer, 129 mer, or 130 mer.

[0038] (Tag or label) Examples of the "tags" included in the assist probe include nucleic acids containing or consisting of a polyA sequence, a polyT sequence, a polyU sequence, a poly(T / U) sequence, a polyG sequence, and a polyC sequence, as well as any specific sequence. The base length of the nucleic acid tag is 5 mer or more and 115 mer or less, 10 mer or more and 110 mer or less, 15 mer or more and 105 mer or less, 20 mer or more and 100 mer or less, 25 mer or more and 95 mer or less, 30 mer or more and 90 mer or less, 35 mer or more and 85 mer or less, 40 mer or more and 80 mer or less, 45 mer or more and 75 mer or less, 50 mer or more and 70 mer or less, or 55 mer or more and 65 mer or less, or alternatively, 5 mer, 6 mer, 7 mer, 8 mer, 9 mer, 10 mer, 11 mer, 12 mer, 13 mer, 14 mer, 15 mer, 16 mer, 17 mer, 18 mer, 19 mer, 20 mer, 21 mer, 22 mer, 23 mer, 24 mer, 25 mer, 26 mer, 27 mer, 28 mer, 29 mer, 30 mer, 31 mer, 32 mer, 33 mer, 34 mer, 35 mer, 36 mer, 37 mer, 38 mer, 39 mer, 40 mer, 41 mer, 42 mer, 43 mer, 44 mer, 45 mer, 46 mer, 47 mer, 48 mer, 49 mer, 50 mer, 51 mer, 52 mer, 53 mer, 54 mer, 55 mer, 56 mer, 57 mer, 58 mer, 59 mer, 60 mer, 61 mer, 62 mer, 63 mer, 64 mer, 65 mer, 66 mer, 67 mer, 68 mer, 69 mer, 70 mer, 71 mer, 72 mer, 73 mer, 74 mer, 75 mer, 76 mer, 77 mer, 78 mer, 79 mer, 80 mer, 81 mer, 82 mer, 83 mer, 84 mer, 85 mer, 86 mer, 87 mer, 88 mer, 89 mer, 90 mer, 91 mer, 92 mer, 93 mer, 94 mer, 95 mer, 96 mer, 97 mer, 98 mer, 99 mer, 100 mer, 101 mer, 102 mer, 103 mer, 104 mer, 105 mer, 106 mer, 107 mer, 108 mer, 109 mer, 110 mer, 111 mer, 112 mer, 113 mer, 114 mer, or 115 mer.In one aspect, the tag or label does not contain nucleic acids such as oligonucleotides or nucleotides. Suitable examples of the "label" included in the assist probe include radioisotopes, biotin, digoxigenin, fluorescent substances, luminescent substances, or dyes. Specifically, 125 I and 32 Radioisotopes such as P, luminescent and chromogenic substances such as digoxigenin and acridinium ester, luminescent substances such as dioxetane, and alkaline phosphatase for using fluorescent substances such as 4-methylumbelliferyl phosphate, and biotin for using fluorescent, luminescent, and chromogenic substances bound to avidin. In addition, it is also possible to detect the target oligonucleotide by adding a donor fluorescent dye and an acceptor fluorescent dye for using fluorescence resonance energy transfer (FRET). In one aspect, the label may be included in another nucleic acid molecule that hybridizes to the nucleic acid tag included in the assist probe. In one aspect, the "label" included in the assist probe is not a radioisotope, biotin, digoxigenin, fluorescent substance, luminescent substance, or dye. In particular, when using biotin, streptavidin, or avidin, and combinations thereof as adapters, in one aspect, the "label" included in the assist probe is not biotin.

[0039] (Adjacent) When the solid phase, tag, or label is "adjacent to", "immobilized on", or "linked to" the nucleotide at the 5'-end or 3'-end of the nucleic acid probe, it primarily means that the solid phase, tag, or label is directly bound to the nucleotide. For example, when the solid phase, tag, or label is bound to the nucleotide via some molecule, the molecule itself can be considered as the solid phase, tag, or label, or it can be considered that the molecule itself constitutes a part of the solid phase, tag, or label. The solid phase may be bound to the nucleic acid probe via an adapter or spacer.

[0040] (Regarding the relationship between the nucleic acid probe - target oligonucleotide and its metabolites) In the present invention, a metabolite refers to an oligonucleotide in which at least one nucleotide is missing from the 3'-end and / or the 5'-end of the target oligonucleotide. In one aspect of the present invention, the metabolite of the target oligonucleotide is missing one or more nucleotides from the 3'-end, and the sequence of the "nucleic acid probe" included in the assist probe is complementary to a partial sequence of the target oligonucleotide including the nucleotide at the 3'-end of the target oligonucleotide, and the sequence of the "nucleic acid probe" included in the capture probe is complementary to a sequence other than the partial sequence of the target oligonucleotide. In this aspect, the tag or label included in the assist probe is adjacent to the nucleotide at the 5'-end of the nucleic acid probe included in the assist probe, and the solid phase included in the capture probe is adjacent to the nucleotide at the 3'-end of the nucleic acid probe included in the capture probe. In another aspect of the present invention, the metabolite of the target oligonucleotide is missing one or more nucleotides from the 5'-end, and the sequence of the "nucleic acid probe" included in the assist probe is complementary to a partial sequence of the target oligonucleotide including the nucleotide at the 5'-end of the target oligonucleotide, and the sequence of the "nucleic acid probe" included in the capture probe is complementary to a sequence other than the partial sequence of the target oligonucleotide. In this aspect, the tag or label included in the assist probe is adjacent to the nucleotide at the 3'-end of the nucleic acid probe included in the assist probe, and the solid phase included in the capture probe is adjacent to the nucleotide at the 5'-end of the nucleic acid probe included in the capture probe. In one aspect of the present invention, it will be naturally understood that the metabolite of the target oligonucleotide missing one or more nucleotides from the 3'-end may be missing one or more nucleotides from the 5'-end, and the metabolite of the target oligonucleotide missing one or more nucleotides from the 5'-end may be missing one or more nucleotides from the 3'-end. Also, in one aspect of the present invention, it will be naturally understood that the sample may include both metabolites of a target oligonucleotide lacking one or more nucleotides from the 3'-end and metabolites of a target oligonucleotide lacking one or more nucleotides from the 5'-end. In this specification, for convenience, the "nucleic acid probe" included in the capture probe may be referred to as the "first nucleic acid probe", and the "nucleic acid probe" included in the assist probe may be referred to as the "second nucleic acid probe". When the first nucleic acid probe included in the capture probe and the second nucleic acid probe included in the assist probe hybridize to the target oligonucleotide, they may be adjacent to each other (without a gap), or may not be adjacent (with a gap of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides). In one aspect of the present invention, when the first nucleic acid probe included in the capture probe and the second nucleic acid probe included in the assist probe hybridize to the target oligonucleotide, they are not adjacent with a gap of 1 to 21 nucleotides, 1 to 16 nucleotides, 1 to 11 nucleotides, or 1 to 7 nucleotides. As another aspect when there is a gap between the first nucleic acid probe included in the capture probe and the second nucleic acid probe included in the assist probe, a blocking probe that is adjacent to each of the first nucleic acid probe included in the capture probe and the second nucleic acid probe included in the assist probe and hybridizes to the gap site of the target oligonucleotide may be used. In this aspect, it will be understood by those skilled in the art that there may be a gap between the first nucleic acid probe and the blocking probe and / or between the second nucleic acid probe and the blocking probe.

[0041] (Regarding complementary - capture probe) When the nucleic acid probe contained in the capture probe is "complementary" to the sequence on the 3'-side (5'-side) of the target oligonucleotide, it preferably means that the sequence of the nucleic acid probe is completely complementary to the sequence of consecutive nucleotides including the nucleotide at the 3'-end (5'-end) of the target oligonucleotide. The length of this completely complementary sequence is preferably the same as the base length of the nucleic acid probe contained in the capture probe. However, in one aspect, in addition to the portion that is completely complementary to the sequence on the 3'-side (5'-side) of the target oligonucleotide, the nucleic acid probe can have additional nucleotides at the 5'-end (3'-end). It will be readily understood that for such additional nucleotides, there is no partner on the target oligonucleotide with which to form a pair or mismatch. The additional nucleotides can also be considered to constitute part or all of a solid phase or an adapter or a spacer adjacent to the nucleotide at the 5'-end (3'-end) of the nucleic acid probe. Also, in one aspect, it will be understood by those skilled in the art that artificial mutations can be introduced into the sequence of the nucleic acid probe, provided that the nucleic acid probe can preferentially bind to the target oligonucleotide compared to the metabolite. Read the content in parentheses as appropriate.

[0042] (Regarding complementary - assist probe) When a nucleic acid probe included in an assist probe is "complementary" to the sequence on the 5'-side (3'-side) of a target oligonucleotide, it preferably means that the sequence of the nucleic acid probe is completely complementary to the sequence of consecutive nucleotides including the nucleotide at the 5'-end (3'-end) of the target oligonucleotide. The length of this completely complementary sequence is preferably the same as the base length of the nucleic acid probe included in the assist probe. However, in one aspect, in addition to the portion that is completely complementary to the sequence on the 5'-side (3'-side) of the target oligonucleotide, the nucleic acid probe can have additional nucleotides at the 3'-end (5'-end). It will be easily understood that there is no partner on the target oligonucleotide with which the additional nucleotides should form a pair or mismatch. The additional nucleotides can also be considered to constitute part or all of a tag adjacent to the nucleotide at the 3'-end (5'-end) of the nucleic acid probe. Also, in one aspect, those skilled in the art will understand that artificial mutations can be introduced into the sequence of the nucleic acid probe. Read appropriately for the content in parentheses.

Example

[0043] [Example 1] Examination of the lengths of the capture probe and the assist probe - 1 (Model where the capture probe and the assist probe are adjacent)

[0044] 1. Materials and methods (1) Target nucleic acid PT2 was used as the target nucleic acid to be measured. As metabolite model nucleic acids of the target nucleic acid, nucleic acid PT2-3n-1 (metabolite 3'n-1 form) with a 1-base deletion at the 3'-end and nucleic acid PT2-5n-1 (metabolite 5'n-1 form) with a 1-base deletion at the 5'-end were used. The nucleic acids were synthesized by Nippon Gene Research Institute (HPLC purification grade). The above-mentioned target nucleic acid is in the same general structure as an antisense nucleic acid, which is a type of nucleic acid drug, and is fully S-modified (phosphorothioated). In PT2, 3 bases each from the 5'-end and 3'-end are replaced by LNA. For PT2-3n-1, 3 bases from the 5'-end and 2 bases from the 3'-end are replaced by LNA. For PT2-5n-1, 2 bases from the 5'-end and 3 bases from the 3'-end are replaced by LNA. PT2, PT2-3n-1, and PT2-5n-1 were all prepared at 0.05, 0.1, 1, or 5 ng / ml using nuclease-free water containing 0.01% Tween 20 and used. Also, blank samples without PT2, PT2-3n-1, and PT2-5n-1 were prepared. 〈Base sequence of PT2〉 5'-G(L)^A(L)^G(L)^C^T^G^A^C^T^T^G^A^T(L)^G(L)^5(L)-3' (the base part is SEQ ID NO: 1) 〈Base sequence of PT2-3n-1〉 5'-G(L)^A(L)^G(L)^C^T^G^A^C^T^T^G^A^T(L)^G(L)-3' (the base part is SEQ ID NO: 2) 〈Base sequence of PT2-5n-1〉 5'-A(L)^G(L)^C^T^G^A^C^T^T^G^A^T(L)^G(L)^5(L)-3' (the base part is SEQ ID NO: 3) ※(L)=LNA, 5 is replaced by 5-Methyl-Cytosine, ^ indicates phosphorothioation.

[0045] (2) Preparation of capture probe MicroPlex as the carrier TMCapture probes were prepared by binding each of the capture probes CP-4m-5N, CP-5m-5N, CP-6m-5N2, CP-7m-5N2, CP-8m-5N2, CP-9m-5N, CP-10m-5N, and CP-11m-5N, which have base lengths of 4mer, 5mer, 6mer, 7mer, 8mer, 9mer, 10mer, and 11mer complementary to the 3'-side of PT2, to the NH 2 modification at the 5'-end of Microspheres (Luminex, product number: LC10015-01). (Hereinafter, these capture probes with different lengths may be collectively referred to as "5'CP-LB"). 〈Base sequence of CP-4m-5N〉 5'-(NH2)-G(L)5(L)A(L)T(L)-3' 〈Base sequence of CP-5m-5N〉 5'-(NH2)-G(L)5(L)A(L)T(L)5(L)-3' 〈Base sequence of CP-6m-5N2〉 5'-(NH2)-G(L)CAT(L)CA(L)-3' 〈Base sequence of CP-7m-5N2〉 5'-(NH2)-G(L)CAT(L)CAA(L)-3' 〈Base sequence of CP-8m-5N2〉 5'-(NH2)-G(L)CATCAAG(L)-3' 〈Base sequence of CP-9m-5N〉 5'-(NH2)-GCATCAAGT-3' 〈Base sequence of CP-10m-5N〉 5'-(NH2)-GCATCAAGTC-3' (The base part is SEQ ID NO: 4) 〈Base sequence of CP-11m-5N〉 5'-(NH2)-GCATCAAGTCA-3' (The base part is SEQ ID NO: 5) ※(L)=LNA, indicating that it is replaced by 5=5-Methyl-Cytosine.

[0046] (3) Capture of target nucleic acid by capture probe (1 st Hybridization reaction) To 10 μL of the target nucleic acid, metabolite model nucleic acid of the target nucleic acid, or blank sample, 25 μL of the 1st Hybridization reaction solution was added to make a total of 35 μL, and the mixture was reacted at 25°C for 1 hour. (3-1)1 st Composition of the Hybridization reaction solution 0.4 μL (800 pieces) of the capture probe immobilized on the carrier in (2) above, 10.5 μL of 5M TMAC (tetramethylammonium chloride), 5.25 μL of 10× supplement [500 mM Tris-HCl (pH 8.0), 40 mM EDTA (pH 8.0), 8.0% sodium N-lauroyl sarcosinate], 5 μL of 17.5% PEG8000 (polyethylene glycol), 2.85 μL of RNase Free water, 1 μL of 100 fmol / ml assist probe (AP-4m, AP-5m, AP-6m, AP-7m', AP-8m', AP-9m', AP-10m', AP-11m' having a base sequence with a polyA chain added to the 3' end of the base sequence complementary to the 5' side of PT2) 〈Base sequence of AP-4m〉 5'-G(L)5(L)T(L)5(L)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 6) 〈Base sequence of AP-5m〉 5'-A(L)G(L)5(L)T(L)5(L)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 7) 〈Base sequence of AP-6m〉 5'-5(L)A(L)G(L)5(L)T(L)5(L)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 8) 〈Base sequence of AP-7m'〉 5'-T(L)CAG(L)CT5(L)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 9) 〈Base sequence of AP-8m'〉 5'-GTCAGCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 10) 〈Base sequence of AP-9m'〉 5'-AGTCAGCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 11) 〈Base sequence of AP-10m'〉 5'-AAGTCAGCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 12) 〈Base sequence of AP-11m'〉 5'-G(L)A(L)A(L)G(L)T(L)5(L)A(L)G(L)5(L)T(L)5(L)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 13) ※(L)=LNA, 5 indicates substitution with 5-Methyl-Cytosine. Hereinafter, these assist probes with different lengths may be collectively referred to as "3'AP-tag".

[0047] (4) Signal amplification by PALSAR reaction To 35 μL of the reaction solution after the 1st hybridization reaction, 15 μL of the PALSAR reaction solution was added to make a total of 50 μL, and the reaction was carried out at 25 °C for 1 hour. The sequences of the pair of self-assembling probes (also referred to as signal amplification probes) used were the following HCP-1 and HCP-2 with their 5'-ends labeled with biotin. 〈Base sequence of HCP-1〉 5'-(Biotin)-CAACAATCAGGACGATACCGATGAAGTTTTTTTTTTTTTTTTTTTT-3' (The base part is SEQ ID NO: 14) 〈Base sequence of HCP-2〉 5'-(Biotin)-GTCCTGATTGTTGCTTCATCGGTATCAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 15) (4-1) Composition of the PALSAR reaction solution Nuclease-Free Water 4.425 μL, 5M TMAC 4.5 μL, 10× supplement [500 mM Tris-HCl (pH8.0), 40 mM EDTA (pH8.0), 8% Sodium N-lauroylsarcosine] 2.75 μL, 20 pmol / μL HCP-1 1.75 μL, 20 pmol / μL HCP-2 1.575 μL

[0048] (5) Fluorescence detection The reaction solution after the PALSAR reaction was washed once with 1xPBS-TP [1xPBS [137 mM Sodium Chloride, 8.1 mM Disodium Phosphate, 2.68 mM Potassium Chloride, 1.47 mM Potassium Dihydrogenphosphate], 0.02% Tween20, 1.5 ppm ProClin300]. Subsequently, 50 μL of the detection reagent [SA-PE (Streptavidin-R-Phycoerythrin, manufactured by Prozyme) 5 μg / mL] was added, and the mixture was allowed to stand for 1 hour at 25°C in the dark. Then, it was washed twice with 1xPBS-TP. Subsequently, 75 μL of 1xPBS-TP was added, and the fluorescence of the beads and the SA-PE conjugate was measured using a Luminex System (manufactured by Luminex), and the signals of the target nucleic acid and the metabolite model nucleic acid were detected.

[0049] (6) Results Table 1 shows the results of cross-reactivity when measuring the target nucleic acid and the metabolite model of the target nucleic acid using capture probes (hereinafter referred to as CP) and assist probes (hereinafter referred to as AP) of each chain length. Gap(mer) in the table indicates the number of bases in the target nucleic acid region not recognized by CP and AP. As shown in Table 1, in each combination of CP chain lengths of 5mer, 6mer, 7mer, 8mer, 9mer, 10mer, 11mer and AP chain lengths of 10mer, 9mer, 8mer, 7mer, 6mer, 5mer, 4mer, the cross-reactivity with the metabolite 5'n-1 form showed less than 1%. Furthermore, by using CP and AP with chain lengths of 5 to 10mer, it was shown that regardless of the orientation of CP and AP, it is possible to suppress the cross-reactivity to less than 1% with almost no detection of both metabolites of 3'n-1 form and 5'n-1 form using the same probe.

[0050]

Table 1

[0051] [Example 2] Examination of the lengths of the capture probe and the assist probe - 2 (model where the binding region not recognized by the capture probe and the assist probe is in the target nucleic acid)

[0052] 1. Materials and methods (1) Target nucleic acid PT3 was used as the target nucleic acid to be measured. As metabolite model nucleic acids of the target nucleic acid, nucleic acid PT3-3n-1 (metabolite 3'n-1 form) with a 1-base deletion at the 3' end and nucleic acid PT3-5n-1 (metabolite 5'n-1 form) with a 1-base deletion at the 5' end were used. The nucleic acids were synthesized by the Japan Gene Research Institute (HPLC purification grade). The above target nucleic acid is fully S-modified (phosphorothioated) like the general structure of antisense nucleic acid, which is one type of nucleic acid drug. In PT3, 3 bases each from the 5' end and 3' end are replaced by LNA. Also, for PT3-3n-1, 3 bases from the 5' end and 2 bases from the 3' end are replaced by LNA. For PT3-5n-1, 2 bases from the 5' end and 3 bases from the 3' end are replaced by LNA. PT3, PT3-3n-1, and PT3-5n-1 were all prepared at 0.5, 1, 5, 10, or 20 ng / ml using nuclease-free water containing 0.01% Tween20 and used. Also, blank samples without PT3, PT3-3n-1, and PT3-5n-1 were prepared. 〈Base sequence of PT3〉 5'-G(L)^A(L)^G(L)^C^T^G^A^C^T^T^A^C^A^G^C^G^A^C^T^T^G^A^T(L)^G(L)^5(L)-3' (the base part is SEQ ID NO: 16) 〈Base sequence of PT3-3n-1〉 5'-G(L)^A(L)^G(L)^C^T^G^A^C^T^T^A^C^A^G^C^G^A^C^T^T^G^A^T(L)^G(L)-3' (the base part is SEQ ID NO: 17) 〈Base sequence of PT3-5n-1〉 5'-A(L)^G(L)^C^T^G^A^C^T^T^A^C^A^G^C^G^A^C^T^T^G^A^T(L)^G(L)^5(L)-3' (the base part is SEQ ID NO: 18) ※(L)=LNA, 5 is replaced by 5-Methyl-Cytosine, ^ indicates phosphorothioation.

[0053] (2) Preparation of capture probe MicroPlex as the carrierTM To microspheres (Luminex, product number: LC10015-01), each capture probe CP-5m-5N, CP-6m-5N2, CP-7m-5N2, CP-8m-5N2, CP-9m-5N, CP-10m-5N having a base length of 5mer, 6mer, 7mer, 8mer, 9mer, 10mer complementary to the 3'-side of PT3 was bound via the NH 2 modification at the 5'-end to prepare a capture probe (5'CP-LB). 〈Base sequence of CP-5m-5N〉 5'-(NH2)-G(L)5(L)A(L)T(L)5(L)-3' 〈Base sequence of CP-6m-5N2〉 5'-(NH2)-G(L)CAT(L)CA(L)-3' 〈Base sequence of CP-7m-5N2〉 5'-G(L)CA(L)T(L)5(L)AA(L)-3' 〈Base sequence of CP-8m-5N2〉 5'-(NH2)-G(L)CATCAAG(L)-3' 〈Base sequence of CP-9m-5N〉 5'-(NH2)-GCATCAAGT-3' 〈Base sequence of CP-10m-5N〉 5'-(NH2)-GCATCAAGTC-3' (the base part is SEQ ID NO: 4) ※(L)=LNA, indicating that it is substituted with 5 = 5-Methyl-Cytosine.

[0054] (3) Capture of target nucleic acid by the capture probe (1 st Hybridization reaction) To 10 μL of the target nucleic acid, the metabolite model nucleic acid of the target nucleic acid or the blank sample, 25 μL of the 1st Hybridization reaction solution was added to make a total of 35 μL, and the reaction was carried out at 25 °C for 1 hour. (3-1) 1 st Composition of the Hybridization reaction solution 0.4 μL (800 copies) of the capture probe immobilized on the carrier in (2) above, 10.5 μL of 5M TMAC (tetramethylammonium chloride), 5.25 μL of 10× supplement [500 mM Tris-HCl (pH 8.0), 40 mM EDTA (pH 8.0), 8.0% sodium N-lauroylsarcosinate], 5 μL of 17.5% PEG8000 (polyethylene glycol), 2.85 μL of RNase Free water, 1 μL of 100 fmol / ml assist probe (AP-5m, AP-6m, AP-7m', AP-8m', AP-9m', AP-10m' having a base sequence with a polyA chain added to the 3'-end of the base sequence complementary to the 5'-side of PT3) (3'AP-tag) 〈Base sequence of AP-5m〉 5'-A(L)G(L)5(L)T(L)5(L)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 7) 〈Base sequence of AP-6m〉 5'-5(L)A(L)G(L)5(L)T(L)5(L)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 8) 〈Base sequence of AP-7m'〉 5'-T(L)CAG(L)CT5(L)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 9) 〈Base sequence of AP-8m'〉 5'-GTCAGCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 10) 〈Base sequence of AP-9m'〉 5'-AGTCAGCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 11) 〈Base sequence of AP-10m'〉 5'-AAGTCAGCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 12) ※(L)=LNA, indicating that it is replaced by 5-Methyl-Cytosine.

[0055] (4) Signal amplification by PALSAR reaction 15 μL of PALSAR reaction solution was added to 35 μL of the reaction solution after the 1st Hybridization reaction to make a total of 50 μL, and the reaction was carried out at 25°C for 1 hour. The sequences of the pair of self-assembling probes (also referred to as signal amplification probes) used have the following HCP-1 and HCP-2 with the 5'-end labeled with biotin. 〈Base sequence of HCP-1〉 5'-(Biotin)-CAACAATCAGGACGATACCGATGAAGTTTTTTTTTTTTTTTTTTTT-3' (The base part is SEQ ID NO: 14) 〈Base sequence of HCP-2〉 5'-(Biotin)-GTCCTGATTGTTGCTTCATCGGTATCAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 15) (4-1) Composition of PALSAR reaction solution Nuclease-Free Water 4.425 μL, 5M TMAC 4.5 μL, 10×supplement [500 mM Tris-HCl (pH8.0), 40 mM EDTA (pH8.0), 8% Sodium N-lauroylsarcosine] 2.75 μL, 20 pmol / μL HCP-1 1.75 μL, 20 pmol / μL HCP-2 1.575 μL

[0056] (5) Fluorescence detection After the PALSAR reaction was completed, the reaction solution was washed once with 1xPBS-TP [1xPBS [137 mM Sodium Chloride, 8.1 mM Disodium Phosphate, 2.68 mM Potassium Chloride, 1.47 mM Potassium Dihydrogenphosphate], 0.02% Tween20, 1.5 ppm ProClin300]. Thereafter, 50 μL of the detection reagent [SA-PE (Streptavidin-R-Phycoerythrin, manufactured by Prozyme) 5 μg / mL] was added, and the mixture was allowed to stand for 1 hour at 25°C in the dark. Then, it was washed twice with 1xPBS-TP. Thereafter, 75 μL of 1xPBS-TP was added, and the fluorescence of the beads and the SA-PE conjugate was measured using a Luminex System (manufactured by Luminex), and the signals of the target nucleic acid and the metabolite model nucleic acid were detected.

[0057] (6) Results Using CPs and APs of each chain length, Table 2 shows the results of cross-reactivity when measuring the target nucleic acid and the metabolite model of the target nucleic acid in the case where the binding region not recognized by the CP and the AP is in the target nucleic acid. Gap(mer) in the table indicates the number of bases in the target nucleic acid region not recognized by the CP and the AP. As shown in Table 2, it was shown that even when there is a Gap region between the CP and the AP, cross-reactivity can be significantly suppressed as in the case where there is no Gap region. Furthermore, even when the binding region not recognized by the CP and the AP is in the target nucleic acid, by using CPs and APs with a chain length of 5 to 10 mer, cross-reactivity can be suppressed to less than 1% with the same probe, regardless of the orientation of the CP and the AP, and hardly detect both metabolites of the 3'n-1 body and the 5'n-1 body.

[0058]

Table 2

[0059] [Example 3] Examination of the combination of capture probes and assist probes with a chain length of 5 to 10 mers

[0060] 1. Materials and methods (1) Target nucleic acid Similar to Example 2, PT3 was used as the target nucleic acid to be measured, and as metabolite model nucleic acids of the target nucleic acid, nucleic acid PT3-3n-1 (metabolite 3'n-1 form) with a 1-base deletion at the 3' end and nucleic acid PT3-5n-1 (metabolite 5'n-1 form) with a 1-base deletion at the 5' end were used. The nucleic acids were commissioned for synthesis from Nippon Gene Research Institute (HPLC purification grade). The above target nucleic acids are completely S-modified (phosphorothioated) similar to the general structure of antisense nucleic acids, which are one type of nucleic acid medicine. In PT3, 3 bases each from the 5' end and the 3' end are replaced with LNA. Also, for PT3-3n-1, 3 bases from the 5' end and 2 bases from the 3' end are replaced with LNA. For PT-3-5n-1, 2 bases from the 5' end and 3 bases from the 3' end are replaced with LNA. PT3, PT3-3n-1, and PT3-5n-1 were all prepared at 2, 20, or 50 ng / ml using nuclease-free water containing 0.01% Tween20 and used. Also, blank samples without PT3, PT3-3n-1, and PT3-5n-1 were prepared.

[0061] (2) Preparation of capture probes MicroPlex as the carrier TM To Microspheres (Luminex, product number: LC10015-01), Each capture probe CP-5m-5N, CP-8m-5N2, CP-10m-5N having a base length of 5mer, 8mer, 10mer complementary to the 3' side of PT3 was bound via NH modification at the 5' end of each to prepare capture probes (5'CP-LB). 2 〈Base sequence of CP-5m-5N〉 〈Base sequence of CP-8m-5N2〉 5'-(NH2)-G(L)5(L)A(L)T(L)5(L)-3' 〈Base sequence of CP-8m-5N2〉 5'-(NH2)-G(L)CATCAAG(L)-3' 〈Base sequence of CP-10m-5N〉 5'-(NH2)-GCATCAAGTC-3' (The base part is SEQ ID NO: 4) ※(L)=LNA, indicating that it is substituted with 5 = 5-Methyl-Cytosine.

[0062] (3) Capture of target nucleic acid by capture probe (1 st Hybridization reaction) To 10 μL of target nucleic acid, metabolite model nucleic acid of target nucleic acid or blank sample, 25 μL of 1st Hybridization reaction solution was added to make a total of 35 μL, and the reaction was carried out at 25 °C for 1 hour. (3-1) 1 st Composition of Hybridization reaction solution 0.4 μL (800 pieces) of the capture probe immobilized on the carrier in (2) above, 10.5 μL of 5M TMAC (tetramethylammonium chloride), 5.25 μL of 10× supplement [500 mM Tris-HCl (pH 8.0), 40 mM EDTA (pH 8.0), 8.0% sodium N-lauroyl sarcosinate], 5 μL of 17.5% PEG8000 (polyethylene glycol), 2.85 μL of RNase Free water, 1 μL of 100 fmol / ml assist probe (AP-5m, AP-8m', AP-10m' having a base sequence with a polyA chain added to the 3' end of the base sequence complementary to the 5' side of PT3) (3'AP-tag) 〈Base sequence of AP-5m〉 5'-A(L)G(L)5(L)T(L)5(L)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 7) 〈Base sequence of AP-8m'〉 5'-GTCAGCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 10) 〈Base sequence of AP-10m'〉 5'-AAGTCAGCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 12) ※(L)=LNA, indicating that it is substituted with 5-Methyl-Cytosine.

[0063] (4) Signal amplification by PALSAR reaction 15 μL of PALSAR reaction solution was added to 35 μL of the reaction solution after the 1st Hybridization reaction to make a total of 50 μL, and the reaction was carried out at 25°C for 1 hour. The sequences of the pair of self-assembling probes (also referred to as signal amplification probes) used are the following HCP-1 and HCP-2 with the 5'-end labeled with biotin. 〈Base sequence of HCP-1〉 5'-(Biotin)-CAACAATCAGGACGATACCGATGAAGTTTTTTTTTTTTTTTTTTTT-3' (The base part is SEQ ID NO: 14) 〈Base sequence of HCP-2〉 5'-(Biotin)-GTCCTGATTGTTGCTTCATCGGTATCAAAAAAAAAAAAAAAAAAAA-3' (The base part is SEQ ID NO: 15) (4-1) Composition of PALSAR reaction solution Nuclease-Free Water 4.425 μL, 5M TMAC 4.5 μL, 10× supplement [500 mM Tris-HCl (pH8.0), 40 mM EDTA (pH8.0), 8% Sodium N-lauroylsarcosine] 2.75 μL, 20 pmol / μL HCP-1 1.75 μL, 20 pmol / μL HCP-2 1.575 μL

[0064] (5) Fluorescence detection After the PALSAR reaction ended, the reaction solution was washed once with 1xPBS-TP [1xPBS [137 mM Sodium Chloride, 8.1 mM Disodium Phosphate, 2.68 mM Potassium Chloride, 1.47 mM Potassium Dihydrogenphosphate], 0.02% Tween20, 1.5 ppm ProClin300]. Thereafter, 50 μL of the detection reagent [SA-PE (Streptavidin-R-Phycoerythrin, manufactured by Prozyme) 5 μg / mL] was added, and the mixture was allowed to stand for 1 hour at 25°C in the dark, and then washed twice with 1xPBS-TP. Thereafter, 75 μL of 1xPBS-TP was added, and the fluorescence of the beads and the SA-PE conjugate was measured with a Luminex System (manufactured by Luminex), and the signals of the target nucleic acid and the metabolite model nucleic acid were detected.

[0065] (6) Results Table 3 shows the results of cross-reactivity when various combinations of CP and AP with a chain length of 5 to 10 mer were used to measure the target nucleic acid and the metabolite model of the target nucleic acid. Gap(mer) in the table indicates the number of bases in the target nucleic acid region not recognized by CP and AP. As shown in Table 3, in the combinations of CP and AP chain lengths of 5 to 10 mer, in the combination of CP5mer-AP5mer, which is the combination of CP and AP with the shortest chain length, or in the combination of CP10mer-AP10mer, which is the combination of CP and AP with the longest chain length, or in various combinations of CP and AP chain lengths in 5 to 10 mer such as CP5mer-AP8mer, CP8mer-AP5mer, CP8mer-AP10mer, and CP10mer-AP8mer, the cross-reactivity between the metabolite 3'n-1 form and the 5'n-1 form was less than 1% for both metabolites. From the above results, it was shown that combinations of CP and AP chain lengths that can suppress the cross-reactivity to less than 1% with almost no detection of both the 3'n-1 form and the 5'n-1 form of metabolites with the same probe, regardless of the orientation of CP and AP, can be freely combined with chain lengths of 5 to 10 mer.

[0066] [Table 3]

[0067] [Example 4] Examination of the case where the 5'-end of the assist probe was linked to a tag 1. Materials and methods (1) Target nucleic acid PT2 or PT3 was used as the target nucleic acid to be measured. As metabolite model nucleic acids of the target nucleic acid, nucleic acids PT2-3n-1 or PT3-3n-1 (metabolite 3'n-1 form) with a 1-base deletion at the 3'-end and nucleic acids PT2-5n-1 or PT3-5n-1 (metabolite 5'n-1 form) with a 1-base deletion at the 5'-end were used. The nucleic acids were synthesized by the Japan Gene Research Institute (HPLC purification grade). The above target nucleic acids are completely S-modified (phosphorothioated) in the same manner as the general structure of antisense nucleic acids, which are one type of nucleic acid medicine. In PT2 and PT3, 3 bases each from the 5'-end and 3'-end are replaced with LNA. In addition, for PT2-3n-1 and PT3-3n-1, 3 bases from the 5'-end and 2 bases from the 3'-end are replaced with LNA. For PT2-5n-1 and PT3-5n-1, 2 bases from the 5'-end and 3 bases from the 3'-end are replaced with LNA. PT2, PT3, PT2-3n-1, PT3-3n-1, PT3-5n-1, and PT3-5n-1 were all prepared at 20 ng / ml using nuclease-free water containing 0.01% Tween20. Also, a blank sample containing no above target nucleic acids and metabolite model nucleic acids of the target nucleic acid was prepared.

[0068] (2) Preparation of capture probe MicroPlex as a carrier TM Microspheres (Luminex, product number: LC10015-01) were added with The capture probe CP-5m-3N with a 5-mer base length complementary to the 5'-side of PT3, at its 3'-end NH 2The capture probe was prepared by binding via modification (hereinafter sometimes referred to as "3'CP-LB"). 〈Base sequence of CP-5m-3N〉 5'-A(L)G(L)5(L)T(L)5(L)-(NH2)-3' ※(L)=LNA, indicating that it is substituted with 5-Methyl-Cytosine.

[0069] (3) Capture of target nucleic acid by the capture probe (1 st (Hybridization reaction) To 10 μL of the target nucleic acid, metabolite model nucleic acid of the target nucleic acid, or blank sample, 25 μL of the 1st Hybridization reaction solution was added to make a total of 35 μL, and the mixture was reacted at 25°C for 1 hour. (3-1) 1 st Composition of the Hybridization reaction solution 0.4 μL (800 pieces) of the capture probe immobilized on the carrier in (2) above, 10.5 μL of 5M TMAC (tetramethylammonium chloride), 5.25 μL of 10× supplement [500 mM Tris-HCl (pH 8.0), 40 mM EDTA (pH 8.0), 8.0% sodium N-lauroyl sarcosinate], 5 μL of 17.5% PEG8000 (polyethylene glycol), 2.85 μL of RNase Free water, 1 μL of 100 fmol / ml assist probe (AP-10m'-5A having a base sequence with a polyA chain added to the 5' end of the base sequence complementary to the 3' side of PT2 and PT3) (hereinafter sometimes referred to as "5'AP-tag") 〈Base sequence of AP-10m'-5A〉 5'-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATCAAGTC-3' (The base part is SEQ ID NO: 19) ※(L)=LNA, indicating that it is substituted with 5-Methyl-Cytosine.

[0070] (4) Signal amplification by the PALSAR reaction After the 1st hybridization reaction, 15 μL of the PALSAR reaction solution was added to 35 μL of the reaction solution to make a total of 50 μL, and the reaction was carried out at 25 °C for 1 hour. The sequences of the pair of self - assemblable probes (also referred to as signal amplification probes) used were the following HCP - 1 and HCP - 2 with their 5'-ends labeled with biotin. 〈Base sequence of HCP - 1〉 5'-(Biotin)-CAACAATCAGGACGATACCGATGAAGTTTTTTTTTTTTTTTTTTTT-3'(The base part is SEQ ID NO: 14) 〈Base sequence of HCP - 2〉 5'-(Biotin)-GTCCTGATTGTTGCTTCATCGGTATCAAAAAAAAAAAAAAAAAAAA-3'(The base part is SEQ ID NO: 15) (4 - 1) Composition of the PALSAR reaction solution Nuclease - Free Water 4.6 μL, 5M TMAC 4.5 μL, 10×supplement [500 mM Tris - HCl (pH8.0), 40 mM EDTA (pH8.0), 8% Sodium N - lauroyl sarcosinate] 2.75 μL, 20 pmol / μL HCP - 1 1.75 μL, 20 pmol / μL HCP - 2 1.575 μL

[0071] (5) Fluorescence detection After the PALSAR reaction was completed, the reaction solution was washed once with 1xPBS - TP [1xPBS [137 mM Sodium Chloride, 8.1 mM Disodium Phosphate, 2.68 mM Potassium Chloride, 1.47 mM Potassium Dihydrogenphosphate], 0.02% Tween20, 1.5 ppm ProClin300]. Subsequently, 50 μL of the detection reagent [SA-PE (Streptavidin-R-Phycoerythrin, manufactured by Prozyme) 5 μg / mL] was added, and the mixture was allowed to stand for 1 hour at 25°C in the dark. Then, it was washed twice with 1xPBS-TP. Subsequently, 75 μL of 1xPBS-TP was added, and the fluorescence of the beads and SA-PE conjugate was measured using a Luminex System (manufactured by Luminex), and the signals of the target nucleic acid and the metabolite model nucleic acid were detected.

[0072] (6) Results To show that the inhibitory effect of the cross-reaction with the metabolite is not affected by the orientation of CP and AP, the results of the cross-reactivity when measuring the target nucleic acid and the metabolite model of the target nucleic acid using AP (5'AP-tag) with the 5'-side of AP linked to the tag are shown in Table 4. Gap(mer) in the table indicates the number of bases in the target nucleic acid region not recognized by CP and AP. As shown in Table 4, in both cases where the target nucleic acid was PT2 and PT3, the cross-reactivity between the 3'n-1 form and the 5'n-1 form was less than 1%. From the results of this study using AP (5'AP-tag) with the 5'-side of AP linked to the tag, it was shown that this measurement system, which can suppress the cross-reactivity to less than 1% without detecting almost both metabolites of the 3'n-1 form and the 5'n-1 form with the same probe, is not affected by the orientation of CP and AP.

[0073]

Table 4

Industrial Applicability

[0074] By using the measurement method of the present invention, in the exploratory stage of pharmaceutical development, pharmacokinetics / pharmacodynamics (PK / PD) screening tests, in the non-clinical stage, safety tests, pharmacological tests and pharmacokinetic tests, and in the clinical stage, the drug concentration in animal or human biological samples administered with the drug can be accurately measured without being affected by metabolites.

Claims

1. A method for measuring a target oligonucleotide in a sample by combining a capture probe and an assist probe based on the principle of hybridization, and for distinguishing and measuring a target oligonucleotide that retains its full-length sequence from its metabolite, wherein the capture probe includes a solid phase and a first nucleic acid probe immobilized on the solid phase, the assist probe includes a tag or label and a second nucleic acid probe linked to the tag or label, and the second nucleic acid probe included in the assist probe has a length of 5 bases, 6 bases, 7 bases, 8 bases, or 9 bases, the nucleotide closest to the tag or label among the nucleotides of the second nucleic acid probe forms a base pair with the nucleotide at the 3'-end or 5'-end of the target oligonucleotide, in the metabolite, one or more consecutive nucleotides including the nucleotide at the 3'-end or 5'-end are missing, the second nucleic acid probe can hybridize to a portion including the nucleotide that is missing in the metabolite of the target oligonucleotide, the first nucleic acid probe can hybridize to a portion other than the above-mentioned portion of the target oligonucleotide, the capture probe, the target oligonucleotide, and the assist probe form a complex, method.

2. The method according to claim 1, wherein when measuring a target oligonucleotide in a sample while distinguishing it from a metabolite in which one or more nucleotides are missing from its 3'-end, the second nucleic acid probe included in the assist probe is linked to the tag or label via the nucleotide at the 5'-end.

3. The method according to claim 1, wherein when measuring a target oligonucleotide in a sample while distinguishing it from a metabolite in which one or more nucleotides are missing from its 5'-end, the second nucleic acid probe included in the assist probe is linked to the tag or label via the nucleotide at the 3'-end.

4. A method for detecting a target oligonucleotide in a sample while distinguishing it from a metabolite in which one or more nucleotides are missing from its 3'-end or 5'-end, the method comprising the following steps: (i) Contacting a sample containing a target oligonucleotide or a metabolite lacking one or more nucleotides from its 3'-end or 5'-end with a capture probe for capturing the target oligonucleotide and an assist probe for detecting the target oligonucleotide to form a complex of the capture probe, the target oligonucleotide, and the assist probe; Here, the capture probe includes a solid phase and a first nucleic acid probe immobilized on the solid phase, and the first nucleic acid probe included in the capture probe has a length of 12 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases, 21 bases, 22 bases, 23 bases, 24 bases, or 25 bases; the assist probe includes a tag or label and a second nucleic acid probe linked to the tag or label, and the second nucleic acid probe included in the assist probe has a length of 5 bases, 6 bases, 7 bases, 8 bases, or 9 bases; the sequence of the second nucleic acid probe is complementary to a partial sequence of the target oligonucleotide including the terminal nucleotides of the target oligonucleotide; the partial sequence includes the nucleotides that are missing in the metabolite; the sequence of the first nucleic acid probe is complementary to a sequence other than the partial sequence of the target oligonucleotide, and the tag or label is linked to the terminal nucleotide of the second nucleic acid probe, and the terminal nucleotide of the second nucleic acid probe forms a base pair with the terminal nucleotide of the target oligonucleotide when the target oligonucleotide hybridizes with the second nucleic acid probe; and (ii) Detecting the target oligonucleotide in the sample by detecting the complex.

5. The method according to claim 4, wherein when detecting the target oligonucleotide in the sample by distinguishing it from a metabolite lacking one or more nucleotides from its 3'-end, the second nucleic acid probe is linked to the tag or label via its 5'-terminal nucleotide, and the sequence of the second nucleic acid probe is complementary to a sequence including the 3'-end of the target oligonucleotide.

6. When detecting a target oligonucleotide in a sample by distinguishing it from a metabolite lacking one or more nucleotides from its 5'-end, the second nucleic acid probe is linked to a tag or label via the nucleotide at its 3'-end, and the sequence of the second nucleic acid probe is complementary to the sequence containing the 5'-end of the target oligonucleotide. The method according to claim 4.

7. The sequence of the second nucleic acid probe is complementary to the 3'-side partial sequence of the target oligonucleotide containing the nucleotide at the 3'-end of the target oligonucleotide, the sequence of the first nucleic acid probe is complementary to the sequence other than the 3'-side partial sequence of the target oligonucleotide, and the tag or label is linked to the nucleotide at the 5'-end of the second nucleic acid probe. The method according to claim 4.

8. The sequence of the second nucleic acid probe is complementary to the 5'-side partial sequence of the target oligonucleotide containing the nucleotide at the 5'-end of the target oligonucleotide, the sequence of the first nucleic acid probe is complementary to the sequence other than the 5'-side partial sequence of the target oligonucleotide, and the tag or label is bound to the nucleotide at the 3'-end of the second nucleic acid probe. The method according to claim 4.

9. The method according to claim 1 or 4, wherein the capture probe includes an adapter or spacer between the first nucleic acid probe and the solid phase.

10. The assist probe includes a tag having a base sequence complementary to a part or all of one of a pair of signal amplification probes capable of self-assembling, The method according to claim 1 or 4, further comprising the following steps: (i) adding to the complex a pair of self-assembling signal amplification probes having complementary base sequence regions capable of hybridizing with each other to form a probe polymer bound to the tag of the assist probe contained in the complex; and (ii) detecting the probe polymer.

11. The method according to claim 10, wherein at least one of the pair of self-assembling signal amplification probes contains a poly-T sequence.

12. The method according to claim 10, wherein at least one of the pair of self-assembling signal amplification probes is labeled with a labeling substance.

13. The pair of signal amplification probes capable of self-assembly consists of a first signal amplification probe and a second signal amplification probe, the first signal amplification probe is a nucleic acid probe containing three or more nucleic acid regions, and containing at least nucleic acid region X, nucleic acid region Y, and nucleic acid region Z or a nucleic acid region Z containing a poly T sequence in order from the 5'-terminal side, the second signal amplification probe is a nucleic acid probe containing three or more nucleic acid regions, and containing at least a nucleic acid region X' complementary to the nucleic acid region X, a nucleic acid region Y' complementary to the nucleic acid region Y, and a nucleic acid region Z' complementary to the nucleic acid region Z or a nucleic acid region Z' containing a poly A sequence in order from the 5'-terminal side, and is characterized in that, The method according to claim 10.

14. A detection kit for distinguishing and measuring a target oligonucleotide in a sample from a metabolite lacking one or more nucleotides from its 3'-terminal or 5'-terminal, the kit comprising a capture probe, an assist probe, and a pair of signal amplification probes having complementary base sequence regions capable of hybridizing to each other and capable of forming a probe polymer by self-assembly, the capture probe includes a solid phase and a first nucleic acid probe immobilized on the solid phase, the first nucleic acid probe included in the capture probe has a length of 12 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases, 20 bases, 21 bases, 22 bases, 23 bases, 24 bases or 25 bases, the assist probe includes a tag having a base sequence complementary to a part or all of one of the pair of signal amplification probes and a second nucleic acid probe linked to the tag, and the second nucleic acid probe included in the assist probe has a length of 5 bases, 6 bases, 7 bases, 8 bases, or 9 bases, the sequence of the second nucleic acid probe is complementary to a partial sequence of the target oligonucleotide containing one or more nucleotides from the 3'-terminal or 5'-terminal of the target oligonucleotide that is missing in the metabolite, the sequence of the first nucleic acid probe is complementary to a sequence other than the partial sequence of the target oligonucleotide, and, The tag is linked to the nucleotide at the end of the second nucleic acid probe, and the nucleotide at the end of the second nucleic acid probe forms a base pair with the nucleotide at the end of the target oligonucleotide when the target oligonucleotide hybridizes with the second nucleic acid probe. Detection kit.

15. The detection kit according to claim 14, wherein the first nucleic acid probe includes an adapter or a spacer between the first nucleic acid probe and the solid phase.

16. A detection kit for distinguishing and measuring a target oligonucleotide in a sample from a metabolite lacking one or more nucleotides from its 3'-end, wherein the second nucleic acid probe included in the assist probe is linked to a tag via the nucleotide at its 5'-end. The detection kit according to claim 14.

17. A detection kit for distinguishing and measuring a target oligonucleotide in a sample from a metabolite lacking one or more nucleotides from its 5'-end, wherein the second nucleic acid probe included in the assist probe is linked to a tag via the nucleotide at its 3'-end. The detection kit according to claim 14.

18. The detection kit according to claim 14, wherein at least one of the pair of signal amplification probes is labeled with a labeling substance.

19. The pair of signal amplification probes consists of a first signal amplification probe and a second signal amplification probe. The first signal amplification probe is a nucleic acid probe including at least a nucleic acid region X, a nucleic acid region Y, and a nucleic acid region Z or a nucleic acid region Z including a poly-T sequence in order from the 5'-end side. The second signal amplification probe is a nucleic acid probe including at least a nucleic acid region X' complementary to the nucleic acid region X, a nucleic acid region Y' complementary to the nucleic acid region Y, and a nucleic acid region Z' complementary to the nucleic acid region Z or a nucleic acid region Z' including a poly-A sequence in order from the 5'-end side. The detection kit according to any one of claims 14 to 18.

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