Nucleic acid measurement method

Abstract

The present invention provides a nucleic acid measurement method for measuring a target nucleic acid separately from a metabolite of the target nucleic acid, the nucleic acid measurement method including: a step for hybridizing a fluorescently labeled probe containing a photocrosslinkable artificial nucleic acid with the target nucleic acid to form a target nucleic acid-fluorescently labeled probe complex; a step for irradiating the target nucleic acid-fluorescently labeled probe complex with ultraviolet light to form a covalent bond between the target nucleic acid and the fluorescently labeled probe, thereby forming a target nucleic acid-fluorescently labeled probe bond body; and a step for analyzing the target nucleic acid-fluorescently labeled probe bond body by high-performance liquid chromatography to detect the peak of the target nucleic acid-fluorescently labeled probe bond body from the fluorescence wavelength emitted by the fluorescently labeled probe. This nucleic acid measurement method makes it possible to accurately measure the target nucleic acid separately from the metabolite thereof with the use of high-performance liquid chromatography.

Description

[Technical field]

[0001] The present invention relates to a nucleic acid measurement method. This application claims priority based on Japanese Patent Application No. 2022-191121, filed on November 30, 2022, the contents of which are incorporated herein by reference. [Background technology]

[0002] Nucleic acid drugs, based on oligonucleotide skeletons, are attracting attention as a new drug discovery modality for treating hereditary diseases that have been difficult to target using previous drug discovery methods. Nucleic acid drugs are generally composed of oligonucleic acids in which nucleic acids or modified nucleic acids are linked together by tens of bases, and act directly on the body without being translated into proteins; they are drugs manufactured by chemical synthesis. Gene therapy products are also composed of nucleic acids, but they differ from nucleic acid drugs in that they act by being translated into proteins and are manufactured biologically. Nucleic acid drugs can be broadly divided into those that act on RNA and those that act on proteins. Antisense and siRNA are the drugs that act on RNA, and these account for the majority of nucleic acid drugs that have been put to practical use so far. Aptamers and CpG oligos are the drugs that act on proteins. The mechanism of action of antisense drugs that have been put to practical use can be broadly divided into two types. One type induces RNA degradation in an RNaseH-dependent manner to negatively control the expression of target genes. Structurally, it is called a gapmer, in which sugar-modified nucleic acids are introduced at both ends of the sequence and unmodified DNA is placed in the center. The other type controls splicing by binding to the splicing regulatory site. This type is called SSO (splice-switching oligonucleotide), and controls the expression of target genes positively or negatively by mechanisms such as exon skipping or exon inclusion. For practical application of nucleic acid drugs, it is important to stabilize them in vivo and improve their binding strength to target RNA, and various modified nucleic acid technologies have been developed and applied. Decomposition of oligonucleotides is mainly by hydrolysis by nucleases in vivo, so stabilization of the phosphodiester bond backbone is necessary. Phosphorothioate modification (PS modification), in which the O atom of the phosphodiester moiety is converted to an S atom, is used as a backbone modification for antisense drugs in the clinical development stage. In PS-modified nucleic acids, chemical modifications are often also introduced to the sugar moiety, such as the introduction of a substituent at the 2' position, such as 2'-O-methylated RNA (2'-OMe), 2'-O-methoxyethylated RNA (2'-MOE), and 2'-fluorinated RNA (2'-F), and cross-linked modifications such as LNA (locked nucleic acid), which is a nucleotide in which the 2' and 4' carbons of the ribose are modified with cross-links, are used. Another representative chemical modification is morpholino oligonucleotides, which are widely used by converting phosphodiester bonds to uncharged phosphorodiamidate bonds and introducing morpholine rings instead of ribose. Antisense drugs currently in clinical development are broadly divided into S-oligonucleotides, which use PS-modified nucleic acids for all nucleic acids, and morpholino oligonucleotides, which use morpholino nucleic acids for all nucleic acids. The pharmacokinetic profiles of nucleic acid drugs, which are chemically synthesized products, are evaluated in accordance with the pharmacokinetic concepts and test methods used for small molecule drugs.

[0003] Patent Document 1 describes a method for detecting a target nucleic acid by a sandwich hybridization method using a detection probe that hybridizes with the target nucleic acid and a capture probe fixed to a support, which is capable of detecting the target nucleic acid with high sensitivity without reducing the detection sensitivity (S / N ratio) even when the amount of detection probe used is reduced, and which includes a step of irradiating light to a complex formed by hybridization of the target nucleic acid with the detection probe and / or capture probe to form a covalent bond between the photoreactive group and the nucleic acid base in the target nucleic acid.

[0004] Patent Document 2 describes a method for improving photoligation efficiency by overcoming the problem of stagnation in photoligation with a target nucleic acid using a probe containing photoresponsive nucleic acids, in which a target site present in a nucleic acid sample is hybridized with a first probe having a sequence complementary to the target site and containing photoresponsive nucleic acids in a reaction solution and then photoligated by light irradiation, the method being characterized in that self-association caused by the photoresponsive nucleic acids in the first probe is suppressed.

[0005] Patent Document 3 describes a method for photocrosslinking by forming a duplex with a photocrosslinkable artificial nucleic acid probe and a target base sequence in an RNA molecule as a means for detecting a base sequence in a difficult-to-detect site in a higher-order structure RNA in a cell, the method including a step of forming a duplex with a photocrosslinkable artificial nucleic acid assist probe and an RNA molecule to perform photocrosslinking simultaneously with or prior to the duplex formation between the photocrosslinkable artificial nucleic acid probe and the target base sequence in the RNA molecule. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2014 / 034753 [Patent Document 2] International Publication No. 2014 / 014106 [Patent Document 3] JP 2022-039526 A Summary of the Invention [Problem to be solved by the invention]

[0007] High performance liquid chromatography (HPLC) is one of the most popular analytical techniques used for qualitative and quantitative analysis. HPLC is capable of rapid and simultaneous qualitative and quantitative analysis of multiple solute components contained in a sample, and because it is composed of a group of highly accurate instruments, it also has excellent reproducibility of analytical values. In addition, the amount of sample injected into HPLC can be measured from the order of a few μL, and the configuration and parameters of the HPLC instrument can be selected according to the analyst's purpose, resulting in a wide range of sample applicability. Due to these characteristics, HPLC has established its position as a scientific technology that supports the foundations of all scientific fields and industries around the world. However, in analyzing the pharmacokinetic properties of nucleic acid drugs that use oligonucleotides as their basic structure, it is necessary to measure the target nucleic acid, which is a nucleic acid drug, separately from its metabolites. However, even with the separation performance of HPLC, it can be difficult to distinguish and accurately measure the target nucleic acid and its metabolites.

[0008] An object of the present invention is to provide a nucleic acid measurement method that can distinguish a target nucleic acid from its metabolites with high accuracy using high performance liquid chromatography. [Means for solving the problem]

[0009] The present invention includes the following aspects. [1] A method for measuring a target nucleic acid by distinguishing it from a metabolite of the target nucleic acid, comprising the steps of: hybridizing a fluorescently labeled probe containing a photocrosslinkable artificial nucleic acid with the target nucleic acid to form a target nucleic acid-fluorescently labeled probe complex; forming a target nucleic acid-fluorescently labeled probe conjugate by irradiating the target nucleic acid-fluorescently labeled probe complex with ultraviolet light to form a covalent bond between the target nucleic acid and the fluorescently labeled probe; and A step of analyzing the target nucleic acid-fluorescently labeled probe conjugate by high performance liquid chromatography and detecting a peak of the target nucleic acid-fluorescently labeled probe conjugate from the fluorescence wavelength emitted by the fluorescent probe. A nucleic acid measurement method comprising the steps of: [2] The nucleic acid measurement method according to [1], wherein the strand length of the fluorescently labeled probe is 4 to 11 mer. [3] The nucleic acid measurement method according to [1] or [2], wherein the fluorescently labeled probe contains two or more artificial nucleic acids. [4] The nucleic acid measurement method according to [3], wherein the artificial nucleic acid is at least one selected from the group consisting of locked nucleic acid, 5-methylcytidylic acid, and 2'-deoxy-5-methylcytidylic acid. [5] The nucleic acid measurement method according to any one of [1] to [4], wherein the target nucleic acid is contained in a sample which is blood or plasma collected from a human, a mouse, a rat, or a monkey. [6] The nucleic acid measurement method according to any one of [1] to [5], wherein the concentration of the target nucleic acid is measured from a peak area of ​​the target nucleic acid and a peak area of ​​an internal standard nucleic acid detected with a fluorescent dye different from that of the target nucleic acid. [7] The nucleic acid measurement method according to any one of [1] to [6], wherein the photocrosslinkable artificial nucleic acid is CNV-K or CNV-D. [8] The method for measuring a nucleic acid according to any one of [1] to [7], wherein at least one phosphodiester bond of the target nucleic acid is modified with phosphorothioate. [9] A fluorescently labeled probe comprising a photocrosslinkable artificial nucleic acid, The fluorescently labeled probe has an oligonucleotide chain length of 4 to 11 mer, a GC content of 40% or more, and contains two or more artificial nucleic acids of at least one type selected from the group consisting of locked nucleic acids, 5-methylcytidylic acid, and 2'-deoxy-5-methylcytidylic acid.

[10] The fluorescently labeled probe according to [9], wherein the photocrosslinkable artificial nucleic acid is CNV-K or CNV-D.

[11] A target nucleic acid-fluorescently labeled probe complex formed by hybridizing the fluorescently labeled probe according to [9] or

[10] with a target nucleic acid.

[12] A target nucleic acid-fluorescently labeled probe conjugate comprising the fluorescently labeled probe according to [9] or

[10] covalently bonded to a target nucleic acid. Effect of the Invention

[0010] According to the present invention, there is provided a nucleic acid measurement method that can distinguish a target nucleic acid from its metabolites and measure it with high accuracy using high performance liquid chromatography. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram for explaining an outline of one embodiment of the present invention. [Diagram 2] FIG. 2 is a graph showing the results of Comparative Example 1. [Diagram 3] FIG. 3 is a graph showing the results of Example 1. [Figure 4] FIG. 4 is a graph showing the results of Comparative Example 2. [Diagram 5] FIG. 5 is a graph showing the results of Example 2. [Figure 6] FIG. 6 is a graph showing the results of Example 3-1. [Figure 7] FIG. 7 is a graph showing the results of Example 3-2. [Figure 8] FIG. 8 is a graph showing the results of Example 3-3. [Figure 9] FIG. 9 is a graph showing the results of Example 3-4. [Figure 10] FIG. 10 is a graph showing the results of Examples 3-5. [Figure 11] FIG. 11 is a graph showing the results of Examples 3-6. [Figure 12] FIG. 12 is a graph showing the results of Examples 3-7. [Figure 13] FIG. 13 is a graph showing the results of Example 4-1. [Figure 14] FIG. 14 is a graph showing the results of Example 4-2. [Figure 15] FIG. 15 is a graph showing the results of Example 4-3. [Figure 16] FIG. 16 is a graph showing the results of Example 5-1. [Figure 17] FIG. 17 is a graph showing the results of Example 5-2. [Figure 18]FIG. 18 is a graph showing the results of Example 5-3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In this specification and claims, when a numerical range is expressed using "~", the numerical range is intended to include the numerical values ​​on both sides of "~".

[0013] [Nucleic acid measurement method] The nucleic acid measurement method of the present invention is a nucleic acid measurement method that measures a target nucleic acid while distinguishing it from metabolites of the target nucleic acid, and includes a complex formation step, a conjugate formation step, and the following steps:

[0014] <Composite formation process> The complex formation step is a step in which a fluorescently labeled probe containing a photocrosslinkable artificial nucleic acid is hybridized with a target nucleic acid to form a target nucleic acid-fluorescently labeled probe complex.

[0015] (target nucleic acid) A target nucleic acid is a nucleic acid that is to be measured by the present invention. The target nucleic acid may be, for example, a nucleic acid drug having an oligonucleic acid as a base skeleton. The oligonucleic acid, which is the backbone of the target nucleic acid, may be modified with phosphorothioate at one or more sites, in which an O atom in the phosphodiester moiety is replaced with an S atom. Furthermore, the oligonucleic acid, which is the basic backbone of the target nucleic acid, may have a substituent introduced at the 2' position of ribose or deoxyribose, such as 2'-O-methylation, 2'-O-methoxyethylation, or 2'-fluorination. Furthermore, the oligonucleic acid, which is the basic backbone of the target nucleic acid, may contain a crosslinked modification such as a locked nucleic acid (LNA). Furthermore, the oligonucleic acid, which is the basic backbone of the target nucleic acid, may contain a morpholino nucleic acid in which the phosphodiester bond is converted to an uncharged phosphorodiamidate bond and a morpholine ring is introduced in place of deoxyribose or ribose.

[0016] The target nucleic acid is preferably contained in a sample which is blood or plasma taken from a human, mouse, rat or monkey.

[0017] The chain length of the target nucleic acid can be appropriately selected depending on the application, etc., and is not particularly limited, but is preferably 10 to 150 mer, more preferably 10 to 50 mer, and even more preferably 10 to 20 mer.

[0018] Non-limiting examples of such nucleic acid drugs include Vitravene (trade name, registered trademark; generic name fomivirsen), Macugen (trade name, registered trademark; generic name pegaptanib), Kynamro (trade name, registered trademark; generic name mipomersen), Exondys 51 (trade name, registered trademark; generic name eteplirsen), Spinraza (trade name, registered trademark; generic name nusinersen), HEPLISAV-B (trade name, registered trademark; generic name CpG1018), Tegsedi (trade name, registered trademark; generic name inotersen), Onpattro (trade name, registered trademark; generic name patisiran), Waylivra (trade name, registered trademark; generic name volanesorsen), Givlaari (trade name, registered trademark; generic name givosiran), Vyondys 53 (trade name, registered trademark; generic name golodirsen), Viltepso (trade name, registered trademark; generic name viltolarsen), and Oxlumo (trade name, registered trademark; generic name Examples include lumasiran.

[0019] (Fluorescently labeled probe) The fluorescently labeled probe can hybridize with the above-mentioned target nucleic acid to form a complex, and includes a photocrosslinkable artificial nucleic acid.

[0020] The base sequence of the fluorescently labeled probe is complementary to at least a part of the base sequence of the target nucleic acid. It is preferable that the fluorescently labeled probe hybridizes to the 5' or 3' side of the target nucleic acid. As the fluorescently labeled probe, it is preferable to use in combination one that hybridizes to the 5' side of the target nucleic acid and one that hybridizes to the 3' side.

[0021] The fluorescent dye used for labeling the fluorescently labeled probe is not particularly limited and may be appropriately selected from those conventionally used for labeling oligonucleic acids. When the fluorescently labeled probe is used in combination with one that hybridizes to the 5' side of the target nucleic acid and one that hybridizes to the 3' side, it is preferable to use a combination of probes with different fluorescent colors. Non-limiting examples of the fluorescent dye include DyLight 405 (excitation wavelength 400 nm, fluorescence wavelength 420 nm), DY-405 (excitation wavelength 400, fluorescence wavelength 420 nm), Alexa Fluor 405 (excitation wavelength 401 nm, fluorescence wavelength 421 nm), AMCA (excitation wavelength 353 nm, fluorescence wavelength 440 nm), AMCA-X (excitation wavelength 347 nm, fluorescence wavelength 447 nm), Pacific Blue (excitation wavelength 405 nm, fluorescence wavelength 455 nm), DY-415 (excitation wavelength 418 nm, fluorescence wavelength 467 nm), Royal Blue (excitation wavelength 426 nm, fluorescence wavelength 480 nm), ATTO 425 (excitation wavelength 436 nm, fluorescence wavelength 484 nm), Cy2 (excitation wavelength 489 nm, fluorescence wavelength 505 nm), ATTO 465 (excitation wavelength 453 nm, emission wavelength 508 nm), DY-475XL (excitation wavelength 492 nm, emission wavelength 509 nm), NorthernLights 493 (excitation wavelength 493 nm, emission wavelength 514 nm), DY-490 (excitation wavelength 490 nm, emission wavelength 516 nm), DyLight 488 (excitation wavelength 493 nm, emission wavelength 518 nm), Alexa Fluor 488 (excitation wavelength 495 nm, fluorescence wavelength 519 nm), 5-FITC (excitation wavelength 494 nm, fluorescence wavelength 519 nm), 5-FAM (excitation wavelength 495 nm, fluorescence wavelength 520 nm), DY-495-X5 (excitation wavelength 495 nm, fluorescence wavelength 520 nm), DY-495 (excitation wavelength 493 nm, fluorescence wavelength 521 nm), Fluorescein (excitation wavelength 494 nm, fluorescence wavelength 521 nm), FITC (excitation wavelength 498 nm, fluorescence wavelength 522 nm), ATTO 488 (excitation wavelength 501 nm, fluorescence wavelength 523 nm), HiLyte Flour 488 (excitation wavelength 499 nm, fluorescence wavelength 523 nm), MFP488 (excitation wavelength 501 nm, fluorescence wavelength 523 nm), ATTO 495 (excitation wavelength 495 nm, fluorescence wavelength 527 nm), Oyster 500 (excitation wavelength 505-530nm), Spectrum Green 497-538nm), ATTO 520 (excitation wavelength 525nm, emission wavelength 545nm), ATTO 532 (excitation wavelength 532nm, emission wavelength 553nm), DY-500XL (excitation wavelength 505nm, emission wavelength 555nm), DY-485XL (excitation wavelength 485nm,Fluorescence wavelength 560nm), Alexa Fluor 555 (excitation wavelength 555nm, fluorescence wavelength 565nm), HiLyte Plus 555 (excitation wavelength 552nm, fluorescence wavelength 567nm), DyLight 549 (excitation wavelength 550nm, fluorescence wavelength 568nm), HiLyte Fluor 555 (excitation wavelength 553nm, fluorescence wavelength 568nm), Cy3 (excitation wavelength 550nm, fluorescence wavelength 570nm), DyLight 547 (excitation wavelength 557nm, emission wavelength 570nm), Rhodamine (excitation wavelength 550nm, emission wavelength 570nm), TRITC (excitation wavelength 550nm, emission wavelength 570nm), DY-548 (excitation wavelength 558nm, emission wavelength 572nm), DY-554 (excitation wavelength 551nm, emission wavelength 572nm), DY-555 (excitation wavelength 547nm, emission wavelength 572nm), Alexa Fluor 546 (excitation wavelength 556nm, emission wavelength nm, emission wavelength 573nm), DY-556 (excitation wavelength 548nm, emission wavelength 573nm), NorthernLights 557 (excitation wavelength 557nm, emission wavelength 574nm), Oyster 550 (excitation wavelength 555 nm, fluorescence wavelength 574 nm), 5-TAMRAnm, fluorescence wavelength 547 (excitation wavelength 574 nm), DY-505-X5 (excitation wavelength 505 nm, fluorescence wavelength 574 nm), DY-547 (excitation wavelength 557 nm, fluorescence wavelength 574 nm), Oyster 556 (excitation wavelength 562 nm, fluorescence wavelength 575 nm), DY-549 (excitation wavelength 560 nm, fluorescence wavelength 575 nm), ATTO 550 (excitation wavelength 554nm, fluorescence wavelength 576nm), PE (excitation wavelength 488nm, fluorescence wavelength 578nm), B-PE (excitation wavelength 545nm, fluorescence wavelength 578nm), R-PE (excitation wavelength 566nm, fluorescence wavelength 578nm), DY-560 (excitation wavelength 559nm, fluorescence wavelength 578nm), TAMRA (excitation wavelength 555nm, fluorescence wavelength 580nm), MFP555 (excitation wavelength 560nm, fluorescence wavelength 585nm), Spectrum Orange (excitation wavelength 559nm, fluorescence wavelength 588nm), DY-510XL (excitation wavelength 509nm, fluorescence wavelength 590nm), ATTO 565 (excitation wavelength 563nm, fluorescence wavelength 592nm), Cy3.5 (excitation wavelength 581nm, fluorescence wavelength 596nm), ROX (X-Rhodamine,Rhodamine Red X) (excitation wavelength 587 nm, fluorescence wavelength 599 nm), DY-590 (excitation wavelength 580 nm, fluorescence wavelength 599 nm), 5-ROX (excitation wavelength 573 nm, fluorescence wavelength 602 nm), Spectrum Red (excitation wavelength 587 nm, fluorescence wavelength 612 nm), ECD (excitation wavelength 488 nm, fluorescence wavelength 613 nm), Texas Red (excitation wavelength 596 nm, fluorescence wavelength 615 nm), DyLight 594 (excitation wavelength 593 nm, fluorescence wavelength 618 nm), Alexa Fluor 594 (excitation wavelength 590 nm, fluorescence wavelength 619 nm), HiLyte Fluor TR (excitation wavelength 591 nm, fluorescence wavelength 622 nm), ATTO 590 (excitation wavelength 594nm, fluorescence wavelength 624nm), MFP590 (excitation wavelength 597nm, fluorescence wavelength 624nm), DY-610 (excitation wavelength 610nm, fluorescence wavelength 630nm), DY-480XL (excitation wavelength 500nm, fluorescence wavelength 630nm), ATTO 610 (excitation wavelength 615nm, fluorescence wavelength 634nm), DY-615 (excitation wavelength 621nm, fluorescence wavelength 641nm), C-PC (C-Phycocyanin) (excitation wavelength 616nm, fluorescence wavelength 647nm), ATTO 620 (excitation wavelength 619nm, fluorescence wavelength 643nm), Phycocyanin (excitation wavelength 620nm, fluorescence wavelength 650nm), DY-481XL (excitation wavelength 515nm, fluorescence wavelength 650nm), ATTO 633 (excitation wavelength 629 nm, fluorescence wavelength 657 nm), DY-630 (excitation wavelength 636 nm, fluorescence wavelength 657 nm), DY-632 (excitation wavelength 637 nm, fluorescence wavelength 657 nm), DY-633 (excitation wavelength 637 nm, fluorescence wavelength 657 nm), MFP631 (excitation wavelength 633 nm, fluorescence wavelength 658 nm), DyLight 633 (excitation wavelength 638 nm, fluorescence wavelength 658 nm), NorthernLights 637 (excitation wavelength 637nm, fluorescence wavelength 658nm), DY-631 (excitation wavelength 637nm, fluorescence wavelength 658nm), DY-634 (excitation wavelength 635nm, fluorescence wavelength 658nm), APC (Allophycocyanin) (excitation wavelength 650nm, fluorescence wavelength 660nm), APC-XL (excitation wavelength 650nm, fluorescence wavelength 662nm), DY-520XL (excitation wavelength 520nm, fluorescence wavelength 664nm), Alexa Fluor 647 (excitation wavelength 650nm,Fluorescence wavelength 665nm), Cy5 (excitation wavelength 643nm, fluorescence wavelength 667nm), DY-521XL (excitation wavelength 523nm, fluorescence wavelength 668nm), Oyster 645 (excitation wavelength 650nm, fluorescence wavelength 669nm), Quantum Red (excitation wavelength 488nm, fluorescence wavelength 670nm), Tri-Color (excitation wavelength 488nm, fluorescence wavelength 670nm), DY-635 (excitation wavelength 647nm, fluorescence wavelength 671nm), DY-636 (excitation wavelength 645nm, fluorescence wavelength 671nm), DY-647 (excitation wavelength 653nm, fluorescence wavelength 672nm), DyLight 647 (excitation wavelength 652nm, fluorescence wavelength 673nm), HiLyte Fluor 647 (excitation wavelength 653nm, fluorescence wavelength 673nm), DyLight 649 (excitation wavelength 646nm, fluorescence wavelength 674nm), HiLyte Plus 647 (excitation wavelength 649nm, fluorescence wavelength nm, fluorescence wavelength 674nm), Oyster 650 (excitation wavelength 655nm, fluorescence wavelength 674nm), DY-648 (excitation wavelength 653nm, fluorescence wavelength 674nm), DY-650 (excitation wavelength 653nm, fluorescence wavelength 674nm), PerCP (excitation wavelength 488nm, fluorescence wavelength 675nm), DY-652 (excitation wavelength 654nm, fluorescence wavelength 675nm), DY-649 (excitation wavelength 655nm, fluorescence wavelength 676nm), DY-651 (excitation wavelength 656nm, fluorescence wavelength 678nm), Oyster 656 (excitation wavelength 662nm, fluorescence wavelength 679nm), ATTO 655 (excitation wavelength 663 nm, fluorescence wavelength 684 nm), Cy5.5 (excitation wavelength 675 nm, fluorescence wavelength 694 nm), DY-677 (excitation wavelength 673 nm, fluorescence wavelength 694 nm), DY-678 (excitation wavelength 674 nm, fluorescence wavelength 698 nm), HiLyte Fluor 680 (excitation wavelength 678 nm, fluorescence wavelength 699 nm), DY-675 (excitation wavelength 674 nm, fluorescence wavelength 699 nm), DY-676 (excitation wavelength 674 nm, fluorescence wavelength 699 nm), IRDye700DX (excitation wavelength 689 nm, fluorescence wavelength 700 nm), DY-681 (excitation wavelength 691 nm, fluorescence wavelength 708 nm), DY-680 (excitation wavelength 690 nm, fluorescence wavelength 709 nm), DY-682 (excitation wavelength 690 nm, fluorescence wavelength 709 nm), DyLight 680 (excitation wavelength 682 nm,Fluorescence wavelength 715nm), Alexa Fluor 700 (excitation wavelength 702nm, fluorescence wavelength 723nm), DY-700 (excitation wavelength 707nm, fluorescence wavelength 730nm), DY-701 (excitation wavelength 706nm, fluorescence wavelength 731nm), PREX710 (excitation wavelength 710nm, fluorescence wavelength 740nm), DY-730 (excitation wavelength 732nm, fluorescence wavelength 758nm), DY-732 (excitation wavelength 736nm, fluorescence wavelength 759nm), DY-734 (excitation wavelength 736nm, fluorescence wavelength 759nm), DY-731 (excitation wavelength 736nm, fluorescence wavelength 760nm), DY-752 (excitation wavelength 748nm, fluorescence wavelength 772nm), DY-750 (excitation wavelength 747nm, fluorescence wavelength 776nm), DyLight 750 (excitation wavelength 752nm, fluorescence wavelength 778nm), HiLyte Fluor 750 (excitation wavelength 754nm, fluorescence wavelength 778nm), DY-749 (excitation wavelength 752nm, fluorescence wavelength 778nm), HiLyte Plus 750 (excitation wavelength 751nm, fluorescence wavelength 779nm), DY-751 (excitation wavelength 751nm, fluorescence wavelength 779nm), DyLight 800 (excitation wavelength 770 nm, fluorescence wavelength 794 nm), IRDye800CW (excitation wavelength 774 nm, fluorescence wavelength 800 nm), DY-780 (excitation wavelength 782 nm, fluorescence wavelength 800 nm), DY-781 (excitation wavelength 783 nm, fluorescence wavelength 800 nm), DY-782 (excitation wavelength 784 nm, fluorescence wavelength 800 nm), DY-776 (excitation wavelength 771 nm, fluorescence wavelength 801 nm), DY-777 (excitation wavelength 771 nm, fluorescence wavelength 801 nm), and IRDye800 (excitation wavelength 778 nm, fluorescence wavelength 806 nm).

[0022] The photocrosslinkable artificial nucleic acid is not particularly limited as long as it can form a crosslink with the base of the target nucleic acid upon irradiation with ultraviolet light. Non-limiting examples of the photocrosslinkable artificial nucleic acid include CNV-K (formula (1) below) with a deoxyribose backbone and CNV-D (formula (2) below) with a D-threoninol backbone (both manufactured by NICCA Chemical Co., Ltd.). Note that formulas (1) and (2) below are shown as phosphoramidites.

[0023] [ka]

[0024] The fluorescently labeled probe preferably contains two or more artificial nucleic acids in addition to the photocrosslinkable artificial nucleic acid. Non-limiting examples of the artificial nucleic acid include locked nucleic acid (LNA), 5-methylcytidylic acid, and 2'-deoxy-5-methylcytidylic acid, and at least one selected from the group consisting of these is preferred.

[0025] The length of the fluorescently labeled probe is not particularly limited, but is preferably 4 to 11 mer, and more preferably 5 to 10 mer. When the length is within this range, the probe is more likely to hybridize specifically to the target nucleic acid.

[0026] (Hybridization) In the complex formation step, hybridization of the target nucleic acid with the fluorescently labeled probe can be carried out by, but is not limited to, incubating the target nucleic acid with the fluorescently labeled probe in a buffer solution.

[0027] <Combined body formation process> The conjugate formation process is a process in which the target nucleic acid-fluorescently labeled probe complex formed in the complex formation process is irradiated with ultraviolet light to form a covalent bond between the target nucleic acid and the fluorescently labeled probe, thereby forming a target nucleic acid-fluorescently labeled probe conjugate.

[0028] (Formation of covalent bonds by photocrosslinking) In the conjugate formation step, the covalent bond between the target nucleic acid and the fluorescently labeled probe of the target nucleic acid-fluorescently labeled probe complex is formed by irradiating the target nucleic acid-fluorescently labeled probe complex with ultraviolet light. The wavelength and irradiation time of the ultraviolet light can be appropriately set depending on the type of photocrosslinkable artificial nucleic acid. When the photocrosslinkable artificial nucleic acid is CNV-K or CNV-D, the wavelength of the ultraviolet light is preferably 313 to 380 nm, more preferably 320 to 380 nm, and the irradiation time of the ultraviolet light is preferably 1 to 30 minutes, more preferably 5 to 15 minutes.

[0029] Peak detection process The peak detection step is a step of analyzing the target nucleic acid-fluorescently labeled probe conjugate formed in the conjugate formation step by high performance liquid chromatography (HPLC) and detecting the peak of the target nucleic acid-fluorescently labeled probe conjugate from the fluorescent wavelength emitted by the fluorescent probe.

[0030] The HPLC conditions are not particularly limited and can be set appropriately. The concentration of the target nucleic acid can be determined from the peak area of ​​the target nucleic acid and the peak area of ​​an internal standard nucleic acid detected with a fluorescent dye different from that of the target nucleic acid.

[0031] [Fluorescently labeled probes, complexes, and conjugates] The fluorescently labeled probe, the target nucleic acid, the complex, and the conjugate are as described above. The fluorescently labeled probe of the present invention has an oligonucleotide chain length of 5 to 10 mer, a GC content of 40% or more, and contains at least one artificial nucleic acid selected from the group consisting of locked nucleic acid, 5-methylcytidylic acid, and 2'-deoxy-5-methylcytidylic acid. The photocrosslinkable artificial nucleic acid is preferably CNV-K or CNV-D. In the fluorescently labeled probe that satisfies these conditions, even when the unchanged product coexists with the 5' metabolite and the 3' metabolite, the fluorescently labeled probe corresponding to the deletion side does not bind to the 5' metabolite and the 3' metabolite that are deleted with only one base, and both the fluorescently labeled probes corresponding to the 5' and 3' sides can hybridize only to the unchanged product. The target nucleic acid-fluorescently labeled probe complex of the present invention is formed by hybridizing the above-mentioned fluorescently labeled probe with a target nucleic acid. The target nucleic acid-fluorescently labeled probe conjugate of the present invention is formed by covalently binding the above-mentioned fluorescently labeled probe to a target nucleic acid.

[0032] [Effects] When the unchanged form, 5' metabolite, and 3' metabolite of the target nucleic acid coexist, the difference in the length of the oligonucleotide chains of these three is often only 1 to 2 nucleotides, so that it is difficult to separate them by HPLC as they are. In one embodiment of the present invention, a fluorescently labeled probe that specifically hybridizes to each of the 5' and 3' ends of the target nucleic acid is used, and the target nucleic acid and the fluorescently labeled probe are cross-linked to form a covalent bond, so that the unchanged form, 5' metabolite, and 3' metabolite can be separated and detected stably and accurately. According to Figure 1, a probe labeled with fluorescent dye A is bound to the 5' side of the unchanged substance and a probe labeled with fluorescent dye B is bound to the 3' side, a probe labeled with fluorescent dye B is bound to the 3' side of the 5' metabolite and a probe labeled with fluorescent dye A is bound to the 5' side of the 3' metabolite. When these are separated by HPLC, the fluorescence of fluorescent dye A and fluorescent dye B is measured for the unchanged substance, whereas only one of them is measured for the metabolites. EXAMPLES

[0033] The present invention will be described in more detail below with reference to experimental examples. However, the scope of the present invention is not limited to the experimental examples described below.

[0034] [material] (1) Target nucleic acid (synthesized by Nippon Gene Research Institute, Ltd.) T-PT1-5'a (unaltered 16mer): SEQ ID NO: 1 Nucleic acid sequence (5'→3'):A(L)^G(L)^A(L)^G^C^T^G^A^C^T^T^G^A^T(L)^G(L)^5(L) (2) Simulated metabolites of target nucleic acid (synthesized by Nippon Gene Research Institute, Ltd.) T-PT1-5'a-3n-1 (3'n-1: 15mer): SEQ ID NO: 2 Nucleic acid sequence (5'→3'):A(L)^G(L)^A(L)^G^C^T^G^A^C^T^T^G^A^T(L)^G(L) T-PT1-5n-1 (5'n-1: 15mer): SEQ ID NO: 3 Nucleic acid sequence (5'→3'):G(L)^A(L)^G^C^T^G^A^C^T^T^G^A^T(L)^G(L)^5(L) (3) Internal standard nucleic acid (synthesized by Nippon Gene Research Institute, Ltd.) T-IS-16mer (internal target nucleic acid: 16mer): SEQ ID NO: 4 Nucleic acid sequence (5'→3'): A(L)GCTTCGAGCTAATCG(L) (4) Fluorescently labeled probe (synthesized by Hokkaido System Science Co., Ltd.) 7mer(3L1D)-5'-FAM(3) (for 5'n-1 discrimination, FAM labeling (3' modification), CNV-D insertion probe): SEQ ID NO: 5 Nucleic acid sequence (5'→3'): 5(L)AD5(L)TCT(L)-FAM 6mer(3L1D)-5'-FAM(3) (for 5'n-1 discrimination, FAM labeling (3' modification), CNV-D insertion probe): SEQ ID NO: 6 Nucleic acid sequence (5'→3'): A(L)D5(L)TCT(L)-FAM 5mer(3L1D)-5'-FAM(3) (for discriminating 5'n-1 form, FAM-labeled (3' modified), CNV-D insertion probe): SEQ ID NO: 7 Nucleic acid sequence (5'→3'): D5(L)TCT(L)-FAM ROX-7mer(3L1D)-3' (for 3'n-1 discrimination, ROX-labeled (5'-modified), CNV-D insertion probe): SEQ ID NO: 8 Nucleic acid sequence (5'→3'): ROX-G(L)CAD5(L)AA(L) ROX-8mer(3L1D)-3' (for 3'n-1 discrimination, ROX-labeled (5'-modified), CNV-D insertion probe): SEQ ID NO: 9 Nucleic acid sequence (5'→3'): ROX-G(L)CAD5(L)AA(L)G ROX-8Mer(3L1D)-3' (for 3'n-1 discrimination, ROX-labeled (5'-modified), CNV-K insertion probe): SEQ ID NO: 10 Nucleic acid sequence (5'→3'): ROX-G(L)CAK5(L)AA(L)G ROX-10mer(3L1D)-3' (for 3'n-1 discrimination, ROX-labeled (5'-modified), CNV-D insertion probe): SEQ ID NO: 11 Nucleic acid sequence (5'→3'): ROX-G(L)CAD5(L)AA(L)GTC Cy5-IS-16mer(D) (for internal target nucleic acid, Cy5 labeling (5' modification), CNV-D insertion probe: SEQ ID NO: 12 Nucleic acid sequence (5'→3'): Cy5-5(L)GATTAGCTCGADGCT(L)

[0035] In the nucleic acid sequences, each symbol has the following meaning: A: Deoxyadenylic acid C: Deoxycytidylic acid G: Deoxyguanylic acid T: Deoxythymidylic acid 5: 2'-deoxy-5-methylcytidylic acid (L): Locked Nucleic Acid (indicating that the oxygen atom at the 2' position of the ribose that constitutes the nucleic acid on the left is bridged with a methylene to the carbon atom at the 4' position) ^: Phosphorothioate modification (indicating that one of the oxygen atoms in the phosphodiester bond has been replaced by a sulfur atom) D:CNV-D (Nicca Chemical Co., Ltd.) K:CNV-K (Nicca Chemical Co., Ltd.) FAM: Fluorescein (excitation wavelength: 494 nm, emission wavelength: 521 nm; green fluorescence) ROX: X-rhodamine (excitation wavelength: 587 nm, emission wavelength: 599 nm; orange fluorescence) Cy5: Sulfonated cyanine dye (excitation wavelength: 643 nm, emission wavelength: 667 nm; red fluorescence)

[0036] [Reagents, reaction solutions, materials, etc.] 10% SDS Solution (SDS Solution, Molecular Biology Grade (10% w / v), Promega, #V6551) Proteinase K (TAKARA, #9034) 20x SSC (Promega, #V4261) 10×supplement: 500mM Tris-HCl / pH8.0 (Thermo Fisher Scientific, #15568-025), 40mM EDTA / pH8.0 (Thermo Fisher Scientific, #15575-038) 10% Tween 20 (CALBIOCHEM) 1% Tween 20 (CALBIOCHEM) 17.5% polyethylene glycol (PEG8000) solution (SIGMA) Nuclease-free water (not DEPC-treated) (Invitrogen, #AM9937) TE buffer (invitrogen,12090015) Tris (Fujifilm Wako Pure Chemical Industries, Ltd., #207-06275) 1 mol / L sodium hydroxide solution (FUJIFILM Wako Pure Chemical Industries, Ltd., #192-02175) Sodium perchlorate (NaClO4) (FUJIFILM Wako Pure Chemical Industries, Ltd., #192-09255) 1mol / L hydrochloric acid (HCl) (FUJIFILM Wako Pure Chemical Industries, Ltd., #083-01095) Bottle Top Filter 1000mL (Thermo Fisher Scientific, #597-4520) BioPro IEX QF anion exchange column (YMC, QF00S05-0546WP) Hard-Shell 96-well skirted PCR plate (Bio-Rad, #HSP9655) RAPID Slit Seal(Meso Scale Discovery,RSS-S96-80122) DNA LoBind tubes, 1.5mL, PCR clean (Eppendorf, #0030108051) Adhesive PCR Plate Seals (Thermo Fisher Scientific, #AB0558) DNA Lobind Microplate 96 / V-PP(Eppendorf,#0030603303)

[0037] [Experimental equipment] Thermo Mixer comfort(Eppendorf) Thermo Mixer F1.5 (Eppendorf) UV irradiation stand (Optocode Co., Ltd., #FL365-SD) HPLC (Shimadzu Corporation, LC2050C) Dry heat sterilizer (Yamato Scientific Co., Ltd., SG400)

[0038] [Experimental Example 1] Peak comparison with and without UV irradiation (Condition A) Hybridization Reaction (1) Preparation of target nucleic acid The target nucleic acid (unmodified form; T-PT1-5'a) was adjusted to 1500 ng / mL using nuclease-free water containing 0.01% Tween 20. The internal standard nucleic acid (internal standard nucleic acid; T-IS-16mer) was also prepared in a similar solution at 3000 ng / mL.

[0039] (2) Preparation of fluorescently labeled probes The FAM5' probe (7mer(3L1D)-5'-FAM(3)) and the Cy5-IS probe (Cy5-IS-16mer(D)) were diluted to 5 μM with TE buffer. (3) Preparation of hybridization buffer For each sample, 5 μL of internal standard nucleic acid (T-IS-16mer), 1.4 μL of FAM5' probe (7mer(3L1D)-5'-FAM(3)) (final concentration: 0.2 μM), 1.4 μL of Cy5-IS probe (Cy5-IS-16mer(D)) (final concentration: 0.2 μM), 8.75 μL of 20×SSC (final concentration: 5×), 5.25 μL of 10×Supplement (final concentration: 1.5×), 4 μL of nuclease-free water containing 17.5% PEG8000 (final concentration: 2%), and 0.35 μL of nuclease-free water containing 1% Tween 20 (final concentration: 0.01%) were mixed and adjusted to a total of 30 μL with TE buffer.

[0040] (4) Hybridization 30 μL of hybridization buffer was added to each 96-well plate (Hard-Shell 96-well skirted PCR plate), and 5 μL of the nucleic acid to be measured was added. The surface was sealed with a plate seal (RAPID Slit Seal), and the plate was centrifuged at 420 × g for 1 minute, and then reacted in a Thermo Mixer comfort at 25 ° C, 800 rpm for 1 hour.

[0041] <Photocrosslinking by UV irradiation> The seal was removed from the plate after the hybridization reaction, and the plate was left standing under an ultraviolet irradiation stand for 10 minutes to allow photocrosslinking. For samples not exposed to UV, the wells were covered with aluminum foil to block UV light.

[0042] <HPLC analysis> (1) Preparation of eluents (solution A and solution B) Eluents A and B were prepared as follows. For solution B, 9.69 g of Tris and 489.76 g of sodium perchlorate were weighed out and dissolved in approximately 3 L of Milli-Q water, and the pH was adjusted to 8.1 with 1 M HCl. Then, the solution was made up to 4 L with milli-Q water, filtered through a bottle-top filter with a pore size of 0.2 μm, and degassed by ultrasonic treatment for 15 minutes. Solution A is solution B without sodium perchlorate, and the method is the same as that for solution B except that the addition of sodium perchlorate is omitted. Composition: Solution A 20mM Tris-HCl, pH8.1 Solution B 20mM Tris-HCl, 1M NaClO4, pH8.1

[0043] (2) Start analysis Using the eluents (Solution A and Solution B) prepared in (1), a column (BioPro IEX QF anion exchange column) was attached, the plate was set in an HPLC device (LC2050C), and analysis was performed using the following method. Separation mode: Anion exchange ·Eluent: Solution A, 20mM Tris-HCl, pH8.1 Solution B, 20mM Tris-HCl, 1M NaClO4, pH8.1 Method: 20~50% B(0~7.5min) 50~90% B(7.5~8.5min) 90% B(8.5~10.5min) 20% B(10.5~14.5min) ·Flow rate: 0.5mL / min

[0044] <Experimental Results> An experimental example in which UV irradiation was not performed is a comparative example (Comparative Example 1), and an experimental example in which UV irradiation was performed is an embodiment (Example 1). The results of Comparative Example 1 are shown in FIG. 2, and the results of Example 1 are shown in FIG. By using a fluorescently labeled probe containing a photocrosslinkable artificial nucleic acid, the chromatographic peak could be effectively detected (Example 1). However, when UV irradiation was not performed, the target nucleic acid and the fluorescently labeled probe were not covalently bonded, and therefore dissociated during HPLC separation, making it impossible to detect the target nucleic acid (Comparative Example 1).

[0045] [Experimental Example 2] Peak comparison with and without UV irradiation (Condition B) Hybridization Reaction (1) Preparation of target nucleic acid The target nucleic acid (unmodified form; T-PT1-5'a) was adjusted to 1500 ng / mL using nuclease-free water containing 0.01% Tween 20. The internal standard nucleic acid (internal standard nucleic acid; T-IS-16mer) was also prepared in a similar solution at 3000 ng / mL.

[0046] (2) Preparation of fluorescently labeled probes The FAM 5' probe (7mer(3L1D)-5'-FAM(3)), ROX 3' probe (ROX-7mer(3L1D)-3'), and Cy5-IS probe (Cy5-IS-16mer(D)) were diluted to 5 μM in TE buffer.

[0047] (3) Preparation of hybridization buffer Per sample, 5 μL of internal standard nucleic acid (T-IS-16mer), 1.4 μL of FAM5' probe (7mer(3L1D)-5'-FAM(3)) (final concentration: 0.2 μM), 1.4 μL of ROX3' probe (ROX-7mer(3L1D)-3') (final concentration: 0.2 μM), 1.4 μL of Cy5-IS probe (Cy5-IS-16mer(D)) (final concentration: 0.2 μM), 8.75 μL of 20×SSC (final concentration: 5×), 5.25 μL of 10×Supplement (final concentration: 1.5×), 4 μL of nuclease-free water containing 17.5% PEG8000 (final concentration: 2%), and 4 μL of nuclease-free water containing 1% Tween 20. 0.35 μL (final concentration: 0.01%) was mixed and adjusted to a total volume of 30 μL with TE buffer.

[0048] (4) Hybridization 30 μL of hybridization buffer was added to each 96-well plate (Hard-Shell 96-well skirted PCR plate), and 5 μL of the nucleic acid to be measured was added. The surface was sealed with a plate seal (RAPID Slit Seal), and the plate was centrifuged at 420 × g for 1 minute, and then reacted in a Thermo Mixer comfort at 25 ° C, 800 rpm for 1 hour.

[0049] <Photocrosslinking by UV irradiation> The seal was removed from the plate after the hybridization reaction, and the plate was left standing under an ultraviolet irradiation stand for 10 minutes to allow photocrosslinking. For samples not exposed to UV, the wells were covered with aluminum foil to block UV light.

[0050] <HPLC analysis> (1) Preparation of eluents (solution A and solution B) Eluents A and B were prepared as follows. To prepare solution B, 40 mL of 1 mol / L sodium hydroxide solution and 489.76 g of sodium perchlorate were weighed out and dissolved in approximately 3 L of Milli-Q water. The solution was then made up to 4 L with Milli-Q water, filtered through a bottle-top filter with a pore size of 0.2 μm, and degassed by ultrasonic treatment for 15 minutes. Solution A is solution B without sodium perchlorate, and the method is the same as that for solution B except that the addition of sodium perchlorate is omitted. Composition: Solution A 10mM NaOH Solution B 10mM NaOH, 1M NaClO4

[0051] (2) Start analysis Using the eluent prepared in (1), a column (BioPro IEX QF anion exchange column) was attached, the plate was set in an HPLC device (LC2050C), and analysis was performed using the following method. Separation mode: Anion exchange ·Eluent: Solution A, 10mM NaOH Solution B, 10mM NaOH, 1M NaClO4 Method: 20~90% B(0~25min) 20% B(25~30min) ·Flow rate: 0.5mL / min

[0052] <Experimental Results> An experimental example in which UV irradiation was not performed is a comparative example (Comparative Example 2), and an experimental example in which UV irradiation was performed is an embodiment (Example 2). The results of Comparative Example 2 are shown in FIG. 4, and the results of Example 2 are shown in FIG. By using a fluorescently labeled probe containing a photocrosslinkable artificial nucleic acid, the chromatographic peak could be effectively detected (Example 2). However, when UV irradiation was not performed, the target nucleic acid and the fluorescently labeled probe were not covalently bonded, and therefore dissociated during HPLC separation, making it impossible to detect the target nucleic acid (Comparative Example 2).

[0053] [Experimental Example 3] Measurement of unchanged substance and metabolites by hybrid specificity Hybridization Reaction (1) Preparation of target nucleic acid The target nucleic acid (unchanged; T-PT1-5'a), the 3' metabolite of the target nucleic acid (T-PT1-5'a-3n-1), and the 5' metabolite of the target nucleic acid (T-PT1-5n-1) were each adjusted to 1500 ng / mL using nuclease-free water containing 0.01% Tween 20. The internal standard nucleic acid (internal standard nucleic acid; T-IS-16mer) was also prepared in a similar solution at 3000 ng / mL.

[0054] (2) Preparation of probe The FAM5' probe (5mer(3L1D)-5'-FAM(3)), ROX3' probe (ROX-7mer(3L1D)-3', ROX-8mer(3L1D)-3'), and Cy5-IS probe (Cy5-IS-16mer(D)) were diluted to 5 μM with TE buffer. Similarly, the ROX3' probe (ROX-10mer(3L1D)-3') was diluted to 0.5 μM with TE buffer.

[0055] (3) Preparation of hybridization buffer Each sample contained 5 μL of internal standard nucleic acid, 1.4 μL of FAM 5' probe (5mer (3L1D)-5'-FAM (3)) (final concentration: 0.2 μM), 1.4 μL of ROX 3' probe (1.4 μL of ROX-7mer (3L1D)-3' (final concentration: 0.2 μM), 1.4 μL of ROX-8mer (3L1D)-3' (final concentration: 0.2 μM), or 1.4 μL of ROX-10mer (3L1D)-3' (final concentration: 0.02 μM), 1.4 μL of Cy5-IS probe (1.4 μL of Cy5-IS-16mer (D)) (final concentration: 0.2 μM), 8.75 μL of 20 × SSC (final concentration: 5 ×), 5.25 μL of 10 × Supplement (final concentration: 1.5 ×), 17.5% 4 μL of nuclease-free water containing PEG8000 (final concentration: 2%) and 0.35 μL of nuclease-free water containing 1% Tween 20 (final concentration: 0.01%) were mixed and adjusted to a total volume of 30 μL with TE buffer.

[0056] (4) Hybridization 30 μL of hybridization buffer was added to each 96-well plate (hard-shell 96-well skirted PCR plate), and 5 μL of the nucleic acid to be measured (unmodified form and metabolite) was added. The surface was sealed with a plate seal (RAPID Slit Seal), and the plate was centrifuged at 420×g for 1 minute, and then reacted in a Thermo Mixer comfort at 25°C and 800 rpm for 1 hour.

[0057] <Photocrosslinking by UV irradiation> The seal was removed from the plate after the hybridization reaction, and the plate was left standing under an ultraviolet irradiation stand for 10 minutes to allow photocrosslinking.

[0058] <HPLC analysis> (1) Preparation of eluents (solution A and solution B) Eluents A and B were prepared as follows. For solution B, 9.69 g of Tris and 489.76 g of sodium perchlorate were weighed out and dissolved in approximately 3 L of Milli-Q water, and the pH was adjusted to 8.1 with 1 M HCl. After that, the solution was made up to 4 L with Milli-Q water, filtered with a bottle-top filter with a pore size of 0.2 μm, and degassed by ultrasonic treatment for 15 minutes. Solution A is solution B without sodium perchlorate, and the method is the same as that for solution B except that the addition of sodium perchlorate is omitted. Composition: Solution A 20mM Tris-HCl, pH8.1 Solution B 20mM Tris-HCl, 1M NaClO4, pH8.1

[0059] (2) Start analysis Using the eluents (Solution A and Solution B) prepared in (1), a column (BioPro IEX QF anion exchange column) was attached, the plate was set in an HPLC device (LC2050C), and analysis was performed using the following method. Separation mode: Anion exchange ·Eluent: Solution A, 20mM Tris-HCl, pH8.1 Solution B, 20mM Tris-HCl, pH8.1, 1M NaClO4 Method: 20~50% B(0~7.5min) 50~90% B(7.5~8.5min) 90% B(8.5~10.5min) 20% B(10.5~14.5min) ·Flow rate: 0.5mL / min

[0060] <Experimental Results> The results of an experimental example (Example 3-1) using the target nucleic acid (unchanged; T-PT1-5'a), 1.4 μL (final concentration: 0.2 μM) of FAM 5' probe (5mer(3L1D)-5'-FAM(3)), and 1.4 μL (final concentration: 0.2 μM) of ROX 3' probe (ROX-7mer(3L1D)-3') are shown in FIG.

[0061] The results of an experimental example (Example 3-2) using the target nucleic acid (5' metabolite; T-PT1-5n-1) and 1.4 μL (final concentration: 0.2 μM) of the FAM 5' probe (5mer(3L1D)-5'-FAM(3)) and 1.4 μL (final concentration: 0.2 μM) of the ROX 3' probe (ROX-7mer(3L1D)-3') are shown in FIG.

[0062] The results of an experimental example (Example 3-3) using the target nucleic acid (3' metabolite; T-PT1-5'a-3n-1) and 1.4 μL (final concentration: 0.2 μM) of FAM 5' probe (5mer(3L1D)-5'-FAM(3)) and 1.4 μL (final concentration: 0.2 μM) of ROX 3' probe (ROX-7mer(3L1D)-3') are shown in FIG.

[0063] The results of an experimental example (Example 3-4) using the target nucleic acid (unchanged; T-PT1-5'a), 1.4 μL (final concentration: 0.2 μM) of FAM 5' probe (5mer(3L1D)-5'-FAM(3)), and 1.4 μL (final concentration: 0.2 μM) of ROX 3' probe (ROX-8mer(3L1D)-3') are shown in Figure 9.

[0064] The results of an experimental example (Example 3-5) using the target nucleic acid (3' metabolite; T-PT1-5'a-3n-1) and 1.4 μL (final concentration: 0.2 μM) of FAM 5' probe (5mer(3L1D)-5'-FAM(3)) and 1.4 μL (final concentration: 0.2 μM) of ROX 3' probe (ROX-8mer(3L1D)-3') are shown in FIG.

[0065] The results of an experimental example (Example 3-6) using the target nucleic acid (unchanged; T-PT1-5'a), 1.4 μL (final concentration: 0.2 μM) of FAM 5' probe (5mer(3L1D)-5'-FAM(3)), and 1.4 μL (final concentration: 0.02 μM) of ROX 3' probe (ROX-10mer(3L1D)-3') are shown in FIG.

[0066] The results of an experimental example (Example 3-7) using the target nucleic acid (3' metabolite; T-PT1-5'a-3n-1) and 1.4 μL (final concentration: 0.2 μM) of FAM 5' probe (5mer(3L1D)-5'-FAM(3)) and 1.4 μL (final concentration: 0.02 μM) of ROX 3' probe (ROX-10mer(3L1D)-3') are shown in FIG.

[0067] In Experimental Example 3-1 (an experiment using an unchanged target nucleic acid), as shown in FIG. 6, both FAM and ROX peaks were detected.

[0068] In Experimental Example 3-2 (an experimental example using a 5' metabolite of a target nucleic acid), as shown in FIG. 7, only a ROX peak was detected, and no FAM peak was detected.

[0069] In Experimental Example 3-3 (an experimental example using a 3' metabolite of a target nucleic acid), as shown in FIG. 8, only a FAM peak was detected, and no ROX peak was detected.

[0070] In Example 3-4 (an experimental example using an unchanged target nucleic acid), as shown in FIG. 9, both FAM and ROX peaks were detected.

[0071] In Example 3-5 (an experimental example using a 3' metabolite of a target nucleic acid), as shown in FIG. 10, only a FAM peak was detected, and no ROX peak was detected.

[0072] In Example 3-6 (an experimental example using a target nucleic acid), as shown in FIG. 11, both FAM and ROX peaks were detected.

[0073] In Example 3-7 (an experimental example using a 3' metabolite of a target nucleic acid), as shown in FIG. 12, only a FAM peak was detected, and no ROX peak was detected.

[0074] As can be seen from the above results, the fluorescently labeled probes specific to the unchanged substance (FAM5' probe, ROX3' probe) do not bind to the 5' metabolites and 3' metabolites, which are missing only one base, but both the fluorescently labeled probes corresponding to the 5' and 3' sides hybridize only to the unchanged substance, suggesting that metabolites and the unchanged substance can be accurately distinguished and measured.

[0075] [Experimental Example 4] Measurement of samples prepared using rat plasma Hybridization Reaction (1) Preparation of target nucleic acid The target nucleic acid (unchanged; T-PT1-5'a), the 5' metabolite of the target nucleic acid (T-PT1-5'a-3n-1), and the 3' metabolite of the target nucleic acid (T-PT1-5n-1) were each adjusted to 100 μg / mL using nuclease-free water containing 0.01% Tween 20. The internal standard nucleic acid (internal standard nucleic acid; T-IS-16mer) was also prepared in a similar solution at 10 μg / mL.

[0076] (2) Preparation of probe The FAM 5' probe (5mer(3L1D)-5'-FAM(3)), ROX 3' probe (ROX-8mer(3L1D)-3'), and Cy5-IS probe (Cy5-IS-16mer(D)) were diluted to 50 μM in TE buffer.

[0077] (3) Preparation of spiked plasma The target nucleic acid (unchanged form; T-PT1-5'a), the 5' metabolite of the target nucleic acid (T-PT1-5'a-3n-1), and the 3' metabolite of the target nucleic acid (T-PT1-5n-1), prepared in (1) at 100 μg / mL, were each diluted to 1 μg / mL with pooled rat EDTA plasma.

[0078] (4) Preparation of proteinase treatment solution Direct PCR Lysis Reagent (Tail) 35.5 μL, 10% SDS solution 35.5 μL (final concentration: 2%), Proteinase K 35.5 μL (final concentration: 1 / 20), and 10 μg / mL internal standard nucleic acid (T-IS-16mer) 35.5 μL (final concentration: 0.4 μg / mL) were mixed (total volume 50 μL) to prepare.

[0079] (5) Proteinase treatment method 50 μL of proteinase treatment solution was added to 50 μL of added plasma, and the mixture was reacted at 55° C., 1500 rpm for 1 hour in a thermomixer (Thermo Mixer F1.5).

[0080] (6) SDS precipitation method After adding 20 μL of 3M KCl, the mixture was left to stand on ice for 5 minutes, then centrifuged (10,000×g, 25° C., 5 minutes), and 60 μL of the supernatant was transferred to a 96-well plate (DNA Lobind Microplate 96 / V-PP).

[0081] (7) Preparation of hybridization buffer 16.5 μL of 20×SSC (final concentration: 3×), 0.5 μL of 50 μM FAM5′ probe (5mer(3L1D)-5′-FAM(3)) (final concentration: 0.4 μM), 0.5 μL of 50 μM ROX3′ probe (ROX-8mer(3L1D)-3′) (final concentration: 0.4 μM), and 0.5 μL of 50 μM Cy5-IS probe (Cy5-IS-16mer(D)) (final concentration: 0.4 μM) were mixed and adjusted to a total volume of 50 μL with nuclease-free water containing 0.01% Tween 20.

[0082] (8) Hybridization 50 μL of hybridization buffer was added to a 96-well plate (DNA Lobind Microplate 96 / V-PP). The plate surface was sealed with adhesive PCR plate seals, and the plate was reacted at 95°C for 5 minutes in a thermostatic bath (dry heat sterilizer (SG400)), and then at 25°C, 900 rpm, and reacted for 1 hour in a thermomixer (Thermo Mixer comfort).

[0083] <Photocrosslinking by UV irradiation> The seal of the plate on which the hybridization reaction had been carried out was removed, and the solution was transferred to another 96-well plate (hard-shell 96-well skirted PCR plate) and left to stand for 10 minutes under an ultraviolet irradiation stand for photocrosslinking.

[0084] <HPLC analysis> (1) Preparation of eluents (solution A and solution B) Eluents A and B were prepared as follows. For solution B, 9.69 g of Tris and 489.76 g of sodium perchlorate were weighed out and dissolved in approximately 3 L of Milli-Q water, and the pH was adjusted to 8.1 with 1 M HCl. After that, the solution was made up to 4 L with Milli-Q water, filtered with a bottle-top filter with a pore size of 0.2 μm, and degassed by ultrasonic treatment for 15 minutes. Solution A is solution B without sodium perchlorate, and the method is the same as that for solution B except that the addition of sodium perchlorate is omitted. Composition: Solution A 20mM Tris-HCl, pH8.1 Solution B 20mM Tris-HCl, pH8.1, 1M NaClO4

[0085] (2) Start analysis Using the eluents (Solution A and Solution B) prepared in (1), a column (BioPro IEX QF anion exchange column) was attached, the plate was set in an HPLC device (LC2050C), and analysis was performed using the following method. Separation mode: Anion exchange ·Eluent: Solution A, 20mM Tris-HCl, pH8.1 Solution B, 20mM Tris-HCl, pH8.1, 1M NaClO4 Method: 20~50% B(0~7.5min) 50~90% B(7.5~8.5min) 90% B(8.5~10.5min) 20% B(10.5~14.5min) ·Flow rate: 0.5mL / min

[0086] <Experimental Results> The results of an experimental example (Example 4-1) using the target nucleic acid (unchanged: T-PT1-5'a), 0.5 μL (final concentration: 0.4 μM) of FAM5' probe (5mer(3L1D)-5'-FAM(3)), and 0.5 μL (final concentration: 0.4 μM) of ROX' probe (ROX-8mer(3L1D)-3') are shown in Figure 13.

[0087] The results of an experimental example (Example 4-2) using the target nucleic acid (5' metabolite; T-PT1-5n-1) and 0.5 μL (final concentration: 0.4 μM) of the FAM 5' probe (5mer(3L1D)-5'-FAM(3)) and 0.5 μL (final concentration: 0.4 μM) of the ROX 3' probe (ROX-8mer(3L1D)-3') are shown in FIG.

[0088] The results of an experimental example (Example 4-3) using the target nucleic acid (3' metabolite; T-PT1-5'a-3n-1) and 0.5 μL (final concentration: 0.4 μM) of FAM 5' probe (5mer(3L1D)-5'-FAM(3)) and 0.5 μL (final concentration: 0.4 μM) of ROX 3' probe (ROX-8mer(3L1D)-3') are shown in FIG.

[0089] In Example 4-1 (an experimental example using an unchanged target nucleic acid), as shown in FIG. 13, both FAM and ROX peaks were detected.

[0090] In Example 4-2 (an experimental example using a 5' metabolite of a target nucleic acid), as shown in FIG. 14, only a ROX peak was detected, and no FAM peak was detected.

[0091] In Example 4-3 (an experimental example using a 3' metabolite of a target nucleic acid), as shown in FIG. 15, only a FAM peak was detected, and no ROX peak was detected.

[0092] As can be seen from the above results, even when rat plasma was used as the actual sample, the fluorescently labeled probes specific to the unchanged substance (FAM5' probe, ROX3' probe) did not bind to the 5' metabolites and 3' metabolites, which are missing only one base, and the fluorescently labeled probes corresponding to the missing side both hybridized to the unchanged substance, suggesting that metabolites and the unchanged substance can be accurately distinguished and measured.

[0093] [Experimental Example 5] Measurement using a fluorescently labeled probe with CNV-K inserted Overview of the experiment HPLC analysis was performed in the same manner as in Experimental Example 3, except that ROX-8mer(3L1K)-3' (sequence number 10) was used instead of ROX-8mer(3L1D)-3' (sequence number 9) as the ROX3' probe.

[0094] <Experimental Results> The results of an experimental example (Example 5-1) using the target nucleic acid (unchanged: T-PT1-5'a), 0.5 μL (final concentration: 0.4 μM) of FAM5' probe (5mer(3L1D)-5'-FAM(3)), and 0.5 μL (final concentration: 0.4 μM) of ROX' probe (ROX-8mer(3L1K)-3') are shown in Figure 16.

[0095] The results of an experimental example (Example 5-2) using the target nucleic acid (5' metabolite; T-PT1-5n-1) and 0.5 μL (final concentration: 0.4 μM) of the FAM 5' probe (5mer (3L1D)-5'-FAM (3)) and 0.5 μL (final concentration: 0.4 μM) of the ROX 3' probe (ROX-7mer (3L1K)-3') are shown in FIG.

[0096] The results of an experimental example (Example 5-3) using the target nucleic acid (3' metabolite; T-PT1-5'a-3n-1) and 0.5 μL (final concentration: 0.4 μM) of FAM 5' probe (5mer (3L1D)-5'-FAM (3)) and 0.5 μL (final concentration: 0.4 μM) of ROX 3' probe (ROX-7mer (3L1K)-3') are shown in Figure 18.

[0097] In Example 5-1 (an experimental example using an unchanged target nucleic acid), as shown in FIG. 16, both FAM and ROX peaks were detected.

[0098] In Example 5-2 (an experimental example using a 5' metabolite of a target nucleic acid), as shown in FIG. 17, only a ROX peak was detected, and no FAM peak was detected.

[0099] In Example 5-3 (an experimental example using a 3' metabolite of a target nucleic acid), as shown in FIG. 18, only a FAM peak was detected, and no ROX peak was detected.

[0100] As can be seen from the above results, even when a fluorescently labeled probe containing CNV-K was used, as in Examples 3-1 to 3-3, the fluorescently labeled probes specific to the unchanged substance (FAM5' probe, ROX3' probe) did not bind to the 5' metabolite and 3' metabolite, which are missing only one base, and the fluorescently labeled probes corresponding to the missing side both hybridized to the unchanged substance, and both the fluorescently labeled probes corresponding to the 5' and 3' sides hybridized only to the unchanged substance, suggesting that metabolites and the unchanged substance can be accurately distinguished and measured.

[0101] [Sequence List] The nucleic acid sequences disclosed herein are set forth in the table below.

[0102] [Table 1] [Industrial Applicability]

[0103] The nucleic acid measurement method of the present invention is useful, for example, in the pharmacokinetic analysis of nucleic acid pharmaceuticals.

Claims

1. A method for measuring a target nucleic acid by distinguishing it from a metabolite of the target nucleic acid, comprising the steps of: A step of hybridizing a fluorescently labeled probe containing a photocrosslinkable artificial nucleic acid with the target nucleic acid to form a target nucleic acid-fluorescently labeled probe complex; forming a target nucleic acid-fluorescently labeled probe conjugate by irradiating the target nucleic acid-fluorescently labeled probe complex with ultraviolet light to form a covalent bond between the target nucleic acid and the fluorescently labeled probe; and A step of analyzing the target nucleic acid-fluorescently labeled probe conjugate by high performance liquid chromatography and detecting a peak of the target nucleic acid-fluorescently labeled probe conjugate from the fluorescence wavelength emitted by the fluorescent probe. Equipped with the chain length of the fluorescently labeled probe is 5 to 8 mer; The method for measuring a nucleic acid, wherein the fluorescently labeled probe comprises two or more artificial nucleic acids.

2. The nucleic acid measurement method described in claim 1, wherein the artificial nucleic acid is at least one selected from the group consisting of locked nucleic acid, 5-methylcytidylic acid, and 2'-deoxy-5-methylcytidylic acid.

3. A nucleic acid measurement method described in claim 1 or 2, wherein the target nucleic acid is contained in a sample which is blood or plasma collected from a human, mouse, rat, or monkey.

4. A nucleic acid measurement method as described in claim 1 or 2, in which the concentration of the target nucleic acid is measured from the peak area area of ​​the target nucleic acid and the peak area area of ​​an internal standard nucleic acid detected with a fluorescent dye different from the target nucleic acid.

5. A nucleic acid measurement method described in claim 1 or 2, wherein the photocrosslinkable artificial nucleic acid is CNV-K or CNV-D.

6. A nucleic acid measurement method as described in claim 1 or 2, wherein at least one phosphodiester bond of the target nucleic acid is modified with phosphorothioate.

7. A fluorescently labeled probe comprising a photocrosslinkable artificial nucleic acid, The fluorescently labeled probe has an oligonucleotide chain length of 5 to 8 mer, a GC content of 40% or more, and contains two or more artificial nucleic acids of at least one type selected from the group consisting of locked nucleic acids, 5-methylcytidylic acid, and 2'-deoxy-5-methylcytidylic acid.

8. The fluorescently labeled probe described in claim 7, wherein the photocrosslinkable artificial nucleic acid is CNV-K or CNV-D.

9. A target nucleic acid-fluorescently labeled probe complex formed by hybridizing the fluorescently labeled probe according to claim 7 or 8 with a target nucleic acid.

10. A target nucleic acid-fluorescently labeled probe conjugate comprising the fluorescently labeled probe according to claim 7 or 8 and a target nucleic acid covalently bonded thereto.

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