Nucleic acid measurement method, target nucleic acid-fluorescently labeled probe complex, and target nucleic acid-fluorescently labeled probe conjugate

Abstract

Provided is a nucleic acid measurement method for separately measuring a target nucleic acid conjugate in which a target nucleic acid is modified with a conjugate and a target nucleic acid conjugate metabolite which is a part or the whole of the target nucleic acid to which the conjugate is not bonded. The method comprises: a step in which a fluorescently labeled probe that includes a photocrosslinkable artificial nucleic acid is hybridized with the target nucleic acid to form a target nucleic acid-fluorescently labeled probe complex; a step in which the target nucleic acid-fluorescently labeled probe complex is irradiated with ultraviolet ray to form a covalent bond between the target nucleic acid and the fluorescently labeled probe, whereby a target nucleic acid-fluorescently labeled probe conjugate is formed; and a step in which the target nucleic acid-fluorescently labeled probe conjugate is analyzed by high-performance liquid chromatography and a peak corresponding to the target nucleic acid-fluorescently labeled probe conjugate is detected from a fluorescence wavelength emitted by the fluorescently labeled probe.

Description

Nucleic acid measurement method, target nucleic acid-fluorescently labeled probe complex, and target nucleic acid-fluorescently labeled probe conjugate

[0001] The present invention relates to a nucleic acid measurement method. This application claims priority based on Provisional Application No. 63 / 712,545 filed in the United States on October 28, 2024, the content of which is incorporated herein by reference.

[0002] Nucleic acid drugs, which use oligonucleotides as their basic structure, are attracting attention as a new drug discovery modality for genetic diseases and other conditions that were difficult to target with conventional drug discovery methods. Nucleic acid drugs generally refer to drugs that are composed of oligonucleotides, which are nucleic acids or modified nucleic acids linked together in groups of tens to tens of bases, and that act directly on living organisms without being translated into proteins, and are manufactured by chemical synthesis. Gene therapy products are also composed of nucleic acids, but they differ from nucleic acid drugs in that they act after being translated into proteins and are manufactured biologically. Nucleic acid drugs can be broadly classified into those that act on RNA and those that act on proteins. Those that act on RNA include antisense and siRNA, which account for the majority of nucleic acid drugs that have been put into practical use so far. Those that act on proteins include aptamers and CpG oligos. There are two main types of mechanisms of action for antisense drugs that have been put into practical use. One type negatively regulates the expression of target genes by inducing RNA degradation in an RNaseH-dependent manner. Structurally, it is called a gapmer, with sugar-modified nucleic acids introduced at both ends of the sequence and unmodified DNA in the middle. The other type regulates splicing by binding to splicing regulatory sites. This is called an SSO (splice-switching oligonucleotide), and it positively or negatively regulates the expression of target genes through mechanisms such as exon skipping or exon inclusion. For the practical application of nucleic acid drugs, in vivo stabilization and improved binding affinity to target RNA are important, and various modified nucleic acid technologies have been developed and applied to date. Since the degradation of oligonucleotides is mainly due to hydrolysis by nucleases in the body, stabilization of the phosphate diester bond backbone is necessary. For the modification of the backbone of antisense drugs in the clinical development stage, phosphorothioate modification (PS modification), in which the oxygen atom of the phosphate diester portion is converted to an sulfur atom, is used.In PS-modified nucleic acids, chemical modifications to the sugar portion are often introduced as well. These include the introduction of substituents at the 2' position, such as 2'-O-methylated RNA (2'-OMe), 2'-O-methoxyethylated RNA (2'-MOE), and 2'-fluorinated RNA (2'-F), as well as cross-linking modifications such as LNA (locked nucleic acid). Another representative chemical modification is the widely used morpholino nucleic acid, in which the phosphate diester bond is converted to an uncharged phosphorodiamidate bond and a morpholin ring is introduced instead of ribose. Currently, antisense drugs under clinical development are broadly classified into S-modified 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 chemically synthesized nucleic acid drugs 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 target nucleic acids that can detect target nucleic acids with high sensitivity without reducing detection sensitivity (S / N ratio) even when the amount of detection probe used is reduced. This method is a sandwich hybridization method for detecting target nucleic acids using a detection probe that hybridizes with the target nucleic acid and a capture probe immobilized on a support, wherein at least one nucleic acid base in the nucleic acid molecule of the detection probe and / or capture probe is substituted with a photoreactive group, and the method includes a step of irradiating the complex formed by the hybridization of the target nucleic acid and the detection probe and / or capture probe with light 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 overcoming the problem of stagnation in photobinding with target nucleic acids using probes containing photoresponsive nucleic acids and improving photobinding efficiency. This method involves hybridizing a target site present in a nucleic acid sample with a first probe containing photoresponsive nucleic acids having a complementary sequence to the target site in a reaction solution and then irradiating it with light to perform photobinding, characterized in that self-assembly caused by the photoresponsive nucleic acids within the first probe is suppressed.

[0005] Patent Document 3 describes a method for detecting base sequences located in hard-to-detect regions of higher-order RNA within cells, which involves photocrosslinking a photocrosslinkable artificial nucleic acid probe with a target base sequence in an RNA molecule by forming a double helix, and includes the step of photocrosslinking a photocrosslinkable artificial nucleic acid assist probe with an RNA molecule by forming a double helix simultaneously with or prior to the double helix formation of the photocrosslinkable artificial nucleic acid probe and the target base sequence in the RNA molecule.

[0006] International Publication No. 2014 / 034753, International Publication No. 2014 / 014106, Japanese Patent Publication No. 2022-039526

[0007] High-performance liquid chromatography (HPLC) is one of the most popular analytical techniques used for qualitative and quantitative analysis. HPLC can rapidly and simultaneously qualitatively and quantitatively analyze multiple solute components contained in a sample, and its high-precision instrumentation provides excellent reproducibility of analytical results. Furthermore, it is possible to measure samples from the order of a few microliters, and the instrumentation and parameters of the HPLC can be selected according to the analyst's purpose, thus achieving broad sample applicability. Due to these characteristics, it has established itself as a scientific technology that underpins all scientific fields and industries worldwide. However, in the pharmacokinetic analysis of nucleic acid drugs with oligonucleotides as their basic structure, it is necessary to distinguish the target nucleic acid, which is the nucleic acid drug, from its metabolites (hereinafter sometimes referred to as "base-deficient target nucleic acids"). Even with the separation performance of HPLC, it has sometimes been difficult to accurately distinguish and measure the target nucleic acid and its metabolites.

[0008] Pharmaceutical compounds are sometimes modified to maximize their effectiveness or reduce side effects. Methods of modification include adding another compound to the pharmaceutical compound, attaching particles, or encapsulating the pharmaceutical compound within a spherical structure. The compound used for modification is called a conjugate. Modification is performed not only on small molecule drugs but also on nucleic acid drugs; the modified target nucleic acid is called a target nucleic acid conjugate. There have been cases where it has been difficult to accurately distinguish between target nucleic acid conjugates and target nucleic acids without a conjugate (hereinafter sometimes referred to as "target nucleic acid conjugate metabolites").

[0009] The present invention aims to provide a nucleic acid measurement method that can accurately distinguish and measure target nucleic acid conjugates and their metabolites using high-performance liquid chromatography.

[0010] The present invention includes the following aspects: [1] A nucleic acid measurement method for distinguishing between a target nucleic acid conjugate, in which a target nucleic acid is modified by a conjugate, and a target nucleic acid conjugate metabolite, which is a part or all of the target nucleic acid to which the conjugate is not bound, 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; 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 to form a target nucleic acid-fluorescently labeled probe conjugate; and analyzing the target nucleic acid-fluorescently labeled probe conjugate by high-performance liquid chromatography and detecting the peak of the target nucleic acid-fluorescently labeled probe conjugate from the fluorescence wavelength emitted by the fluorescently labeled probe. [2] The nucleic acid measurement method according to [1], wherein the chain 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 loc 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 conjugate or the target nucleic acid conjugate metabolite is contained in a sample which is blood, plasma, or tissue taken from a human, mouse, rat, or monkey. [6] The nucleic acid measurement method according to any one of [1] to [5], wherein the photocrosslinkable artificial nucleic acid is CNV-K phosphoramidite or CNV-D phosphoramidite. [7] A target nucleic acid-fluorescently labeled probe complex formed by hybridizing a fluorescently labeled probe and a target nucleic acid conjugate. [8] A target nucleic acid-fluorescently labeled probe conjugate formed by covalent bonding of a fluorescently labeled probe and a target nucleic acid conjugate.

[0011] According to the present invention, a nucleic acid measurement method is provided that can distinguish and accurately measure target nucleic acid conjugate metabolites using high-performance liquid chromatography.

[0012] This figure shows an overview of the HPLC analysis of the experimental examples. This figure shows an overview of Experimental Example 1. This figure shows an overview of Experimental Example 2. This is an HPLC chromatogram showing that in Experimental Example 1, the target nucleic acid conjugate and its metabolite (target nucleic acid) were separated and detected. This is an HPLC chromatogram showing that in Experimental Example 2, the metabolite with a missing 3' end of the target nucleic acid was not observed as a peak. This is an HPLC chromatogram showing that in Experimental Example 2, the metabolite with a missing 5' end of the target nucleic acid was not observed as a peak. This is an HPLC chromatogram showing that in Experimental Example 2, the conjugate of the target nucleic acid with a missing 5' end and its metabolite (target nucleic acid with a missing 5' end) were separated and detected.

[0013] In this specification and the claims, when a numerical range is indicated using "~", the numerical range shall include the numbers on both sides of "~".

[0014] [Nucleic Acid Measurement Method] The nucleic acid measurement method of the present invention is a nucleic acid measurement method that distinguishes and measures a target nucleic acid conjugate and a target nucleic acid conjugate metabolite, and comprises a complex formation step, a conjugate formation step, and the following steps.

[0015] <Complex Formation Process> The complex formation process involves hybridizing a fluorescently labeled probe containing a photocrosslinkable artificial nucleic acid with a target nucleic acid to form a target nucleic acid-fluorescently labeled probe complex.

[0016] (Target Nucleic Acid) The target nucleic acid is the nucleic acid to be measured by the present invention. The target nucleic acid may be, for example, a nucleic acid drug with an oligonucleotide as its basic structure. The oligonucleotide, which is the basic structure of the target nucleic acid, may have one or more phosphorothioate modifications in which the oxygen atom of the phosphate diester portion is replaced with an oxygen atom. The oligonucleotide, which is the basic structure of the target nucleic acid, may also have substituents introduced at the 2' position, such as 2'-O-methylation, 2'-O-methoxyethylation, or 2'-fluorination of ribose or deoxyribose. The oligonucleotide, which is the basic structure of the target nucleic acid, may also contain cross-linking modifications such as Locked Nucleic Acid (LNA). The oligonucleotide, which is the basic structure of the target nucleic acid, may also contain morpholino nucleic acid, in which the phosphate diester bond is converted to an uncharged phosphorodiamidate bond and a morpholin ring is introduced instead of deoxyribose or ribose. The oligonucleotide, which is the basic structure of the target nucleic acid, may also be a peptide nucleic acid. Furthermore, the target nucleic acid may be single-stranded or double-stranded. The target nucleic acid also includes metabolites in which the 5' or 3' terminal base is missing.

[0017] (Conjugate) The target nucleic acid of the present invention may be bound to a conjugate. Known conjugates include high molecular weight compounds such as polyethylene glycol, lipids, lipid nanoparticles, peptides, proteins such as antibodies, and sugars such as N-acetyl-D-galactosamine and mannose. In the present invention, the target nucleic acid bound to a conjugate is called a target nucleic acid conjugate. Covalent bonds and hydrogen bonds are examples of binding modes between the target nucleic acid and the conjugate. The conjugate may be bound to the 5' end of the target nucleic acid, to the 3' end, or to both ends. The present invention allows for the measurement of the target nucleic acid conjugate and metabolites of the substance (target nucleic acid conjugate) separately. In the present invention, metabolites include, for example, metabolites in which the conjugate portion is missing as a result of the target nucleic acid conjugate being metabolized (or degraded), metabolites in which the bases at the 5' or 3' end of the target nucleic acid sequence are missing, and metabolites that have undergone both of these metabolites. Here, a metabolite in which only the conjugate portion is missing corresponds to the entire target nucleic acid. Furthermore, metabolites in which one or more bases are missing from the 5' or 3' end of the target nucleic acid sequence, in addition to the conjugate portion, are considered to be part of the target nucleic acid.

[0018] Preferably, at least one of the target nucleic acid conjugate and its metabolites is contained in a sample that is blood, plasma, or tissue collected from a human, mouse, rat, or monkey. The target nucleic acid conjugate can be metabolized by the action of enzymes contained in the biological sample, or by changes in pH or temperature.

[0019] The chain length of the target nucleic acid can be appropriately selected depending on its application and other factors, 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.

[0020] Non-exclusive 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 Examples include 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 lumasiran).

[0021] (Fluorescently labeled probe) The fluorescently labeled probe contains a base sequence capable of hybridizing with the target nucleic acid described above to form a complex, and the base sequence contains a photocrosslinkable artificial nucleic acid.

[0022] The nucleotide sequence of the fluorescently labeled probe is complementary to at least a portion of the nucleotide sequence of the target nucleic acid. The fluorescently labeled probe preferably hybridizes to the 5' or 3' end of the target nucleic acid, and more preferably hybridizes to a nucleotide sequence including the 5' or 3' end of the target nucleic acid. Either one of the fluorescently labeled probes that hybridizes to the 5' or 3' end of the target nucleic acid may be used, or a combination of them may be used.

[0023] The fluorescently labeled probe may have a spacer between the fluorescent dye and the base sequence. The presence of a spacer increases the measurement sensitivity. Examples of spacers include linear divalent linking groups having alkylene glycol units (repeating units). A specific example is the formula: -O(CH ２ CH ２A divalent linking group represented as O)n- is an example. In the above formula, n represents an integer from 1 to 10.

[0024] The fluorescent dye used for labeling the fluorescently labeled probe is not particularly limited and can be appropriately selected from those conventionally used for labeling oligonucleotides. When using a combination of fluorescently labeled probes that hybridize to the 5' side and those that hybridize to the 3' side of the target nucleic acid, it is preferable to use a combination with different fluorescent colors. Non-limiting examples of the fluorescent dyes include DyLight 405 (excitation wavelength 400 nm, fluorescence wavelength 420 nm), DY-405 (excitation wavelength 400 nm, 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, fluorescence wavelength 508 nm), DY-475XL (excitation wavelength 492 nm, fluorescence wavelength 509 nm), NorthernLights 493 (excitation wavelength 493 nm, fluorescence wavelength 514 nm), DY-490 (excitation wavelength 490 nm, fluorescence wavelength 516 nm), DyLight 488 (excitation wavelength 493 nm, fluorescence 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 Fluor 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,(Emission wavelength 530 nm), Spectrum Green 497 (538 nm), ATTO 520 (excitation wavelength 525 nm, fluorescence wavelength 545 nm), ATTO 532 (excitation wavelength 532 nm, fluorescence wavelength 553 nm), DY-500XL (excitation wavelength 505 nm, fluorescence wavelength 555 nm), DY-485XL (excitation wavelength 485 nm, fluorescence wavelength 560 nm), Alexa Fluor 555 (excitation wavelength 555 nm, fluorescence wavelength 565 nm), HiLyte Plus 555 (excitation wavelength 552 nm, fluorescence wavelength 567 nm), DyLight 549 (excitation wavelength 550 nm, fluorescence wavelength 568 nm), HiLyte Fluor 555 (excitation wavelength 553 nm, fluorescence wavelength 568 nm), Cy3 (excitation wavelength 550 nm, fluorescence wavelength 570 nm), DyLight 547 (excitation wavelength 557 nm, fluorescence wavelength 570 nm), Rhodamine (excitation wavelength 550 nm, fluorescence wavelength 570 nm), TRITC (excitation wavelength 550 nm, fluorescence wavelength 570 nm), DY-548 (excitation wavelength 558 nm, fluorescence wavelength 572 nm), DY-554 (excitation wavelength 551 nm, fluorescence wavelength 572 nm), DY-555 (excitation wavelength 547 nm, fluorescence wavelength 572 nm), Alexa Fluor 546 (excitation wavelength 556 nm, fluorescence wavelength 573 nm), DY-556 (excitation wavelength 548 nm, fluorescence wavelength 573 nm), NorthernLights 557 (excitation wavelength 557 nm, fluorescence wavelength 574 nm), Oyster 550 (excitation wavelength 555 nm, fluorescence wavelength 574 nm), 5-TAMRA nm, 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 554 nm, fluorescence wavelength 576 nm), PE (excitation wavelength 488 nm, fluorescence wavelength 578 nm), B-PE (excitation wavelength 545 nm, fluorescence wavelength 578 nm), R-PE (excitation wavelength 566 nm, fluorescence wavelength 578 nm), DY-560 (excitation wavelength 559 nm, fluorescence wavelength 578 nm), TAMRA (excitation wavelength 555 nm, fluorescence wavelength 580 nm), MFP555 (excitation wavelength 560 nm,(Emission wavelength 585 nm), Spectrum Orange (excitation wavelength 559 nm, fluorescence wavelength 588 nm), DY-510XL (excitation wavelength 509 nm, fluorescence wavelength 590 nm), ATTO 565 (excitation wavelength 563 nm, fluorescence wavelength 592 nm), Cy3.5 (excitation wavelength 581 nm, fluorescence wavelength 596 nm), 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 594 nm, fluorescence wavelength 624 nm), MFP590 (excitation wavelength 597 nm, fluorescence wavelength 624 nm), DY-610 (excitation wavelength 610 nm, fluorescence wavelength 630 nm), DY-480XL (excitation wavelength 500 nm, fluorescence wavelength 630 nm), ATTO 610 (excitation wavelength 615 nm, fluorescence wavelength 634 nm), DY-615 (excitation wavelength 621 nm, fluorescence wavelength 641 nm), C-PC (C-Phycocyanin) (excitation wavelength 616 nm, fluorescence wavelength 647 nm), ATTO 620 (excitation wavelength 619 nm, fluorescence wavelength 643 nm), Phycocyanin (excitation wavelength 620 nm, fluorescence wavelength 650 nm), DY-481XL (excitation wavelength 515 nm, fluorescence wavelength 650 nm), 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 637 nm, fluorescence wavelength 658 nm), DY-631 (excitation wavelength 637 nm,Fluorescence wavelength 658 nm), DY-634 (excitation wavelength 635 nm, fluorescence wavelength 658 nm), APC (Allophycocyanin) (excitation wavelength 650 nm, fluorescence wavelength 660 nm), APC-XL (excitation wavelength 650 nm, fluorescence wavelength 662 nm), DY-520XL (excitation wavelength 520 nm, fluorescence wavelength 664 nm), Alexa Fluor 647 (excitation wavelength 650 nm, fluorescence wavelength 665 nm), Cy5 (excitation wavelength 643 nm, fluorescence wavelength 667 nm), DY-521XL (excitation wavelength 523 nm, fluorescence wavelength 668 nm), Oyster 645 (excitation wavelength 650 nm, fluorescence wavelength 669 nm), Quantum Red (excitation wavelength 488 nm, fluorescence wavelength 670 nm), Tri-Color (excitation wavelength 488 nm, fluorescence wavelength 670 nm), DY-635 (excitation wavelength 647 nm, fluorescence wavelength 671 nm), DY-636 (excitation wavelength 645 nm, fluorescence wavelength 671 nm), DY-647 (excitation wavelength 653 nm, fluorescence wavelength 672 nm), DyLight 647 (excitation wavelength 652 nm, fluorescence wavelength 673 nm), HiLyte Fluor 647 (excitation wavelength 653 nm, fluorescence wavelength 673 nm), DyLight 649 (excitation wavelength 646 nm, fluorescence wavelength 674 nm), HiLyte Plus 647 (excitation wavelength 649 nm, fluorescence wavelength nm, fluorescence wavelength 674 nm), Oyster 650 (excitation wavelength 655 nm, fluorescence wavelength 674 nm), DY-648 (excitation wavelength 653 nm, fluorescence wavelength 674 nm), DY-650 (excitation wavelength 653 nm, fluorescence wavelength 674 nm), PerCP (excitation wavelength 488 nm, fluorescence wavelength 675 nm), DY-652 (excitation wavelength 654 nm, fluorescence wavelength 675 nm), DY-649 (excitation wavelength 655 nm, fluorescence wavelength 676 nm), DY-651 (excitation wavelength 656 nm, fluorescence wavelength 678 nm), Oyster 656 (excitation wavelength 662 nm, fluorescence wavelength 679 nm), 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 715 nm), Alexa Fluor 700 (excitation wavelength 702 nm, fluorescence wavelength 723 nm), DY-700 (excitation wavelength 707 nm, fluorescence wavelength 730 nm), DY-701 (excitation wavelength 706 nm, fluorescence wavelength 731 nm), PREX710 (excitation wavelength 710 nm, fluorescence wavelength 740 nm), DY-730 (excitation wavelength 732 nm, fluorescence wavelength 758 nm), DY-732 (excitation wavelength 736 nm, fluorescence wavelength 759 nm), DY-734 (excitation wavelength 736 nm, fluorescence wavelength 759 nm), DY-731 (excitation wavelength 736 nm, fluorescence wavelength 760 nm), DY-752 (excitation wavelength 748 nm, fluorescence wavelength 772 nm), DY-750 (excitation wavelength 747 nm, fluorescence wavelength 776 nm), DyLight 750 (excitation wavelength 752 nm, fluorescence wavelength 778 nm), HiLyte Fluor 750 (excitation wavelength 754 nm, fluorescence wavelength 778 nm), DY-749 (excitation wavelength 752 nm, fluorescence wavelength 778 nm), HiLyte Plus 750 (excitation wavelength 751 nm, fluorescence wavelength nm, fluorescence wavelength 779 nm), DY-751 (excitation wavelength 751 nm, fluorescence wavelength 779 nm), DyLight Examples include the 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).

[0025] The photocrosslinkable artificial nucleic acid is not particularly limited as long as it forms crosslinks with the bases of the target nucleic acid upon ultraviolet irradiation. Non-limiting examples of the photocrosslinkable artificial nucleic acid include CNV-K phosphoramidite (formula (1) below) and CNV-D phosphoramidite (formula (2) below) (both manufactured by Nikka Chemical Co., Ltd.).

[0026]

[0027] 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 roq 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.

[0028] The chain 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 chain length is within this range, the target nucleic acid and the target nucleic acid conjugate are more easily separated by HPLC.

[0029] (Hybridization) In the complex formation process, hybridization of the target nucleic acid and the fluorescently labeled probe can be carried out by incubating the target nucleic acid and the fluorescently labeled probe in a buffer, but is not limited to this. Here, the target nucleic acid to which the fluorescently labeled probe binds may be the target nucleic acid of the target nucleic acid conjugate, the target nucleic acid of the target nucleic acid conjugate metabolite, or both.

[0030] <Conjugate Formation Step> The conjugate formation step is a step in which ultraviolet light is irradiated onto the target nucleic acid-fluorescently labeled probe complex formed in the complex formation step 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.

[0031] (Formation of covalent bonds by photocrosslinking) In the conjugate formation step, covalent bonding between the target nucleic acid and the fluorescently labeled probe in the target nucleic acid-fluorescently labeled probe complex is performed by irradiating the target nucleic acid-fluorescently labeled probe complex with ultraviolet light. The wavelength of the ultraviolet light and the irradiation time can be appropriately set depending on the type of photocrosslinkable artificial nucleic acid. When the photocrosslinkable artificial nucleic acid is CNV-K phosphoramidite, the wavelength of the ultraviolet light is preferably 313 to 380 nm, and more preferably 320 to 380 nm. The irradiation time of the ultraviolet light is preferably 0.5 to 90 minutes, and more preferably 0.5 to 40 minutes.

[0032] <Peak Detection Step> The peak detection step involves analyzing the target nucleic acid-fluorescently labeled probe conjugate formed in the conjugate formation step using high-performance liquid chromatography (HPLC) and detecting the peak of the target nucleic acid-fluorescently labeled probe conjugate from the fluorescence wavelength emitted by the fluorescently labeled probe. The type of column used in HPLC is not particularly limited, and known columns can be applied. For example, an ion exchange column can be used to detect the presence or absence of a conjugate in the target nucleic acid by the difference in ionic intensity, and a size exclusion column can be used to detect it by the difference in molecular weight.

[0033] The HPLC conditions are not particularly limited and can be set as appropriate. The concentration of the target nucleic acid can be 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 different fluorescent dye.

[0034] [Fluorescently labeled probe, complex, and conjugate] The fluorescently labeled probe, target nucleic acid, complex, and conjugate are as described above. The fluorescently labeled probe of the present invention preferably has an oligonucleotide chain length of 5 to 10 mer, a GC content of 10% or more, and contains two or more artificial nucleic acids selected from the group consisting of loc nucleic acid, 5-methylcytidylic acid, and 2'-deoxy-5-methylcytidylic acid. The photocrosslinkable artificial nucleic acid is preferably CNV-K phosphoramidite or CNV-D phosphoramidite. The target nucleic acid-fluorescently labeled probe complex of the present invention may be formed by hybridizing the above-described fluorescently labeled probe and target nucleic acid conjugate. The target nucleic acid-fluorescently labeled probe conjugate of the present invention may be formed by hybridizing the above-described fluorescently labeled probe and target nucleic acid conjugate.

[0035] [Effects] When target nucleic acids, their 5' metabolites, and 3' metabolites coexist, the difference in oligonucleotide chain length between these three is often only one or two nucleotides, making separation by HPLC difficult. In one embodiment of the present invention, a fluorescently labeled probe that specifically hybridizes with the target nucleic acid is used, and by cross-linking and covalently bonding the target nucleic acid and the fluorescently labeled probe, the target nucleic acid and the base-deficient target nucleic acid can be stably and accurately separated and detected. Furthermore, even when the target nucleic acid conjugate and the target nucleic acid conjugate metabolites coexist, they can be separated and detected by the same method. For example, the target nucleic acid conjugate and its metabolites can be distinguished by the difference in ionicity caused by the presence or absence of the conjugate or the base deficiency, and this difference in ionicity can be separated and detected by HPLC.

[0036] The present invention will be described in more detail below with experimental examples, but the scope of the present invention is not limited to the experimental examples described later.

[0037] [Materials] (1) Target nucleic acid (synthesized by commission to Japan Gene Research Institute Co., Ltd.) ・T-PT1-5'a(16mer): Sequence ID 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) Target nucleic acid conjugate (synthesized by commission to Japan Gene Research Institute Co., Ltd.) ・GalNAc-T-PT1-5'a(16mer): Sequence ID 2 Nucleic acid sequence (5'→3'): Gal-A(L)^G(L)^A(L)^G^C^T^G^A^C^T^T^T^G^A^T(L)^G(L)^5(L) (3) Base-deficient metabolite of target nucleic acid (synthesized by commission to Japan Gene Research Institute Co., Ltd.)・T-PT1-5'a-3n-1 (3'n-1 form: 15mer): Sequence ID 3 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 form: 15mer): Sequence ID 4 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) (4) Fluorescently labeled probe (synthesized by commission to Hokkaido System Science Co., Ltd.) ・ROX-8mer (ROX labeled): Sequence ID 5 Nucleic acid sequence (5'→3'): ROX-S18-G(L)CAD5(L)AA(L)G

[0038] In 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 ^: Phosphothioate modification D: CNV-D phosphoramidite (Nikka Chemical Co., Ltd.) ROX: X-Rhodamine (excitation wavelength: 587 nm, fluorescence wavelength: 599 nm; orange fluorescence) Gal: N-acetylgalactosamine (GalNAc) S18: Hexaethylene glycol (spacer with 18 atoms)

[0039] [Reagents] 20x SSC (manufactured by Promega) Acetonitrile (manufactured by FUJIFILM Wako Pure Chemical Corporation) 1M Tris-HCl (pH 8.0) (manufactured by Thermo Fisher Scientific) 500 mM EDTA (pH 8.0) (manufactured by Thermo Fisher Scientific) 10% Tween 20 (manufactured by CALBIOCHEM) Nuclease-free Water (not DEPC-treated) (manufactured by Invitrogen) Sodium perchlorate (NaClO ４ )(manufactured by FUJIFILM Wako Pure Chemical Corporation) DirectPCR Lysis Reagent (Tail) (manufactured by Biagen Biotech) 3M Potassium chloride solution (manufactured by FUJIFILM Wako Pure Chemical Corporation) SDS Solution 10%, w / v (manufactured by Promega) Proteinase K (manufactured by TAKARA) Rat pooled plasma (Jackson Laboratory Japan Co., Ltd.)

[0040] [Equipment] HPLC system: Nexera XS Inert (manufactured by Shimadzu Corporation) Shaking incubator: SIC-320LW (manufactured by AS ONE Corporation) Tube shaker: Thermo Mixer F1.5 (manufactured by Eppendorf) Micro high-speed cooling centrifuge: MDX-310 (manufactured by Tomy Seiko Co., Ltd.) UV irradiation stand: FL365-SD (manufactured by Optcode Corporation)

[0041] [Experimental Example 1] The experiment was conducted according to the outline shown in Figure 2. Details are given below. Measurement of target nucleic acid conjugate and target nucleic acid (target nucleic acid conjugate metabolite) <Hybridization reaction> (1) Preparation of measurement sample Target nucleic acid (T-PT1-5'a) and target nucleic acid conjugate (GalNAc-T-PT1-5'a) were each added to rat plasma and adjusted to a concentration of 300 ng / mL. (2) Preparation of hybridization solution A hybridization solution was prepared by mixing 0.88 μL of ROX-8mer (final concentration: 1.76 μmol / L), 32.62 μL of 0.01% Tween solution, and 16.5 μL of 20×SSC. (3) Pretreatment To 50 μL of the sample to be measured, 50 μL of deproteinization solution (per sample: DirectPCR Lysis Reagent (Tail): 35.5 μL, SDS Solution 10%, w / v: 10 μL, Proteinase K: 2.5 μL, 0.01% Tween 20: 2 μL) was added. Then, the mixture was reacted using a tube shaker at 1500 rpm for 1 hour. After the reaction, 20 μL of 3 M potassium chloride solution was added to the solution and allowed to stand on ice for 5 minutes. Then, the mixture was centrifuged (10,000 g, 25°C, 5 minutes), and 60 μL of the supernatant was transferred to DNA Lobind Microplate 96 / V-PP (Eppendorf). (4) 50 μL of hybridization solution was added to the sample after hybridization pretreatment and allowed to stand in an incubator at 95°C for 5 minutes. Then, the mixture was reacted in a shaking incubator at 25°C and 900 rpm for 1 hour. The entire volume of the reacted sample was transferred to a 96-well plate (Hard-Shell 96-well skirted PCR plate, Bio-Rad).

[0042] <Photocrosslinking by UV irradiation> The seal on the plate that underwent the hybridization reaction was removed, and the plate was left to stand under a UV irradiation stand for 30 minutes to undergo photocrosslinking (wavelength: 365 nm). The sample after photocrosslinking was used for HPLC analysis.

[0043] <HPLC Analysis> HPLC analysis was performed according to the outline shown in Figure 1. Details are given below. (1) Preparation of eluents (Solution A, Solution B) Eluents A and B were prepared according to the following procedure. For eluent A, 25 mL of 1 M Tris-HCl (pH 8.0) and 2 mL of 500 mM EDTA (pH 8.0) were made up to 700 mL with test water, then mixed with 300 mL of acetonitrile, and degassed under reduced pressure using an aspirator for 15 minutes. For eluent B, 250 mL of 2 M sodium perchlorate, 25 mL of 1 M Tris-HCl (pH 8.0), and 2 mL of 500 mM EDTA (pH 8.0) were made up to 700 mL with test water, then mixed with 300 mL of acetonitrile, and degassed under reduced pressure using an aspirator for 15 minutes. Composition: Solution A: Test water containing 1mM EDTA and 25mM Tris-HCl / acetonitrile (7 / 3, v / v) Solution B: 0.5M NaClO ４ (1) Analysis Using the eluents (Solution A, Solution B) prepared in (1), a column (BioPro IEX QF 3μm, 4.6×100mm, YMC Corporation) was mounted, and the plate was set in an HPLC instrument (Nexera XS Inert, Shimadzu Corporation), and the analysis was performed under the following conditions: Separation mode: Anion exchange Eluents: Solution A, 1 mM EDTA and 25 mM Tris-HCl-containing test water / acetonitrile (7 / 3, v / v) Solution B, 0.5 M NaClO ４ , 1 mM EDTA and 25 mM Tris-HCl-containing test water / acetonitrile (7 / 3, v / v) • Gradient method: 60 → 80% B (0-15 min) 80% B (15-18 min) 100% B (18-20 min) 60% B (20-30 min) • Flow rate: 0.6 mL / min • Column temperature: 55°C • Injection volume: 30 μL • Fluorescence detection: ROX (excitation light wavelength: 578 nm, fluorescence wavelength: 604 nm)

[0044] <Results> Using fluorescence emitted from the fluorescence-labeled probe (ROX-8mer) hybridized with the target nucleic acid conjugate (GalNAc-T-PT1-5’a) and the target nucleic acid (T-PT1-5’a) contained in the above measurement sample, measurement was performed using HPLC. The results are shown in Fig. 4.

[0045] From the results in Fig. 4, it was shown that the target nucleic acid conjugate and the target nucleic acid were separated from each other and could be accurately distinguished and measured. The ionic property changes depending on the presence or absence of the conjugate. Therefore, it became possible to accurately distinguish and measure using the anion exchange chromatography used in this embodiment. In this embodiment, the elution time of the target nucleic acid conjugate was earlier than the elution time of the target nucleic acid, and two peaks were separated and detected.

[0046] [Experimental Example 2] The experiment was conducted according to the outline shown in Figure 3. Details are given below. Measurement of target nucleic acid and base-deficient metabolites of target nucleic acid (1) Preparation of measurement samples The 3'n-1 metabolite of the target nucleic acid (T-PT1-5'a-3n-1) or the 5'n-1 metabolite of the target nucleic acid (T-PT1-5n-1) was each prepared to 300 ng / mL using rat plasma. In addition, measurement samples were prepared by mixing the target nucleic acid (T-PT1-5'a) and the 5'n-1 metabolite of the target nucleic acid (T-PT1-5'a-5n-1) in rat plasma to 300 ng / mL each. (2) Preparation of hybridization solution A hybridization solution was prepared by mixing 0.88 μL of ROX-8mer (final concentration: 100 μL), 32.62 μL of 0.01% Tween solution, and 16.5 μL of 20×SSC. (3) Pretreatment 50 μL of deproteinization solution (per sample: DirectPCR Lysis Reagent (Tail): 35.5 μL, SDS Solution 10%, w / v: 10 μL, Proteinase K: 2.5 μL, 0.01% Tween 20: 2 μL) was added to 50 μL of the sample to be measured. The mixture was then reacted using a tube shaker at 1500 rpm for 1 hour. 20 μL of 3 M potassium chloride solution was added to the reaction solution and allowed to stand on ice for 5 minutes. The mixture was then centrifuged (10,000 g, 25°C, 5 minutes), and 60 μL of the supernatant was transferred to DNA Lobind Microplate 96 / V-PP (Eppendorf). (4) 50 μL of hybridization solution was added to the sample after hybridization pretreatment and allowed to stand in an incubator at 95°C for 5 minutes. Then, the mixture was reacted in a shaking incubator at 25°C and 900 rpm for 1 hour. The entire volume of the reacted sample was transferred to a 96-well plate (Hard-Shell 96-well skirted PCR plate, Bio-Rad).

[0047] <Photocrosslinking by UV irradiation> The seal on the plate that underwent the hybridization reaction was removed, and the plate was left to stand under a UV irradiation stand for 30 minutes to undergo photocrosslinking (wavelength: 365 nm). The sample after photocrosslinking was used for HPLC analysis.

[0048] <HPLC Analysis> (1) Preparation of Eluents (Solution A, Solution B) Eluents A and B were prepared according to the following procedure. For Eluent A, 25 mL of 1 M Tris-HCl (pH 8.0) and 2 mL of 500 mM EDTA (pH 8.0) were made up to 700 mL with test water, then mixed with 300 mL of acetonitrile, and degassed under reduced pressure using an aspirator for 15 minutes. For Eluent B, 250 mL of 2 M sodium perchlorate, 25 mL of 1 M Tris-HCl (pH 8.0), and 2 mL of 500 mM EDTA (pH 8.0) were made up to 700 mL with test water, then mixed with 300 mL of acetonitrile, and degassed under reduced pressure using an aspirator for 15 minutes. Composition: Solution A: Test water containing 1mM EDTA and 25mM Tris-HCl / acetonitrile (7 / 3, v / v) Solution B: 0.5M NaClO ４ (1) Analysis Using the eluents (Solution A, Solution B) prepared in (1), a column (BioPro IEX QF 3μm, 4.6×100mm, YMC Corporation) was mounted, and the plate was set in an HPLC instrument (Nexera XS Inert, Shimadzu Corporation), and the analysis was performed under the following conditions: Separation mode: Anion exchange Eluents: Solution A, 1 mM EDTA and 25 mM Tris-HCl-containing test water / acetonitrile (7 / 3, v / v) Solution B, 0.5 M NaClO ４ , 1 mM EDTA and 25 mM Tris-HCl-containing test water / acetonitrile (7 / 3, v / v) • Gradient method: 60 → 80% B (0-15 min) 80% B (15-18 min) 100% B (18-20 min) 60% B (20-30 min) • Flow rate: 0.6 mL / min • Column temperature: 55°C • Injection volume: 30 μL • Fluorescence detection: ROX (excitation light wavelength: 578 nm, fluorescence wavelength: 604 nm)

[0049] <Results> Figure 5 shows the results of measuring the 3'n-1 metabolite of the target nucleic acid (T-PT1-5'a-3n-1) using a fluorescently labeled probe (ROX-8mer). Figure 6 shows the results of measuring the 5'n-1 metabolite of the target nucleic acid (T-PT1-5n-1) using a fluorescently labeled probe (ROX-8mer). Figure 7 shows the results of measuring the target nucleic acid (T-PT1-5'a) and its 5'n-1 metabolite (T-PT1-5n-1) using a fluorescently labeled probe (ROX-8mer).

[0050] When a sample containing the 3'n-1 metabolite of the target nucleic acid was measured using a fluorescently labeled probe (ROX-8mer), no peak was detected (Figure 5). The fluorescently labeled probe (ROX-8mer) has a sequence complementary to the 3' end of the target nucleic acid. Therefore, the 3'n-1 metabolite, which lacks a base at the 3' end, did not hybridize with the fluorescently labeled probe, and no peak was detected. Note that the arrow pointing to "target nucleic acid" in Figure 5 indicates the position where a peak is expected to appear if the "target nucleic acid" is present in the measurement sample.

[0051] When a sample containing the 5'n-1 metabolite of the target nucleic acid was measured using a fluorescently labeled probe (ROX-8mer), a peak was detected (Figure 6). Because the fluorescently labeled probe (ROX-8mer) has a sequence complementary to the 3' end of the target nucleic acid, hybridization is possible even if the 5' end base is missing, resulting in the detection of a peak. The arrow pointing to "target nucleic acid" in Figure 6 indicates the expected location where a peak would appear if the "target nucleic acid" is present in the sample.

[0052] When a sample containing the target nucleic acid and its 5'n-1 metabolite was measured using a fluorescently labeled probe (ROX-8mer), two peaks were detected (Figure 7). The target nucleic acid and its 5'n-1 metabolite retain the 3' terminal base intact. Therefore, the peak was detected by hybridizing with the fluorescently labeled probe (ROX-8mer), which has a complementary sequence to the 3' terminal of the target nucleic acid. Furthermore, in this embodiment, the measurement was performed using the principle of ion exchange chromatography. Due to structural differences, the target nucleic acid and its 5' terminal base-deficient metabolite have different charge distributions, resulting in differences in retention time (elution time). By utilizing this characteristic, it is possible to distinguish between the two and measure them individually.

[0053] Based on these results, this technology makes it possible to accurately distinguish between target nucleic acids, target nucleic acid conjugates, 3'n-1 metabolites of target nucleic acids, and 5'n-1 metabolites of target nucleic acids.

[0054] [Sequence List] The nucleic acid sequences disclosed in this specification are shown in the table below. Note that the sequence described in Sequence ID No. 5 is a sequence consisting of fewer than 10 bases and is therefore not included in the sequence listing.

[0055]

[0056] The nucleic acid measurement method of the present invention is useful, for example, in the pharmacokinetic analysis of nucleic acid drugs.

Claims

1. A nucleic acid measurement method for distinguishing between a target nucleic acid conjugate, in which the target nucleic acid is modified by a conjugate, and a target nucleic acid conjugate metabolite, which is a part or all of the target nucleic acid to which the conjugate is not bound, 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; 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 to form a target nucleic acid-fluorescently labeled probe conjugate; and analyzing the target nucleic acid-fluorescently labeled probe conjugate by high-performance liquid chromatography and detecting the peak of the target nucleic acid-fluorescently labeled probe conjugate from the fluorescence wavelength emitted by the fluorescently labeled probe.

2. The nucleic acid measurement method according to claim 1, wherein the chain length of the fluorescently labeled probe is 4 to 11 mer.

3. The nucleic acid measurement method according to claim 1 or 2, wherein the fluorescently labeled probe comprises two or more artificial nucleic acids.

4. The nucleic acid measurement method according to claim 3, wherein the artificial nucleic acid is at least one selected from the group consisting of loc nucleic acid, 5-methylcytidylic acid, and 2'-deoxy-5-methylcytidylic acid.

5. The nucleic acid measurement method according to claim 1 or 2, wherein the target nucleic acid conjugate or the target nucleic acid conjugate metabolite is contained in a sample which is blood, plasma or tissue taken from a human, mouse, rat, or monkey.

6. The nucleic acid measurement method according to claim 1 or 2, wherein the photocrosslinkable artificial nucleic acid is CNV-K phosphoramidite or CNV-D phosphoramidite.

7. A target nucleic acid-fluorescent-labeled probe complex formed by the hybridization of a fluorescently labeled probe and a target nucleic acid conjugate.

8. A target nucleic acid-fluorescent-labeled probe conjugate formed by the covalent bonding of a fluorescently labeled probe and a target nucleic acid conjugate.

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