Nucleobase detection agent

Abstract

To provide an inexpensive method of easy design and detection, and high-sensitivity mutation detection means capable of detecting even a difference of modification mode.SOLUTION: Provided is a nucleobase detection agent for detecting a predetermined base in a test nucleic acid that includes nucleic acid molecules including a base sequence region hybridizable with the test nucleic acid, wherein the base sequence region includes a detection domain consisting of one acting base, and one display base adjacent to the acting base. The nucleic acid molecule has a constitution in which, when hybridized with the test nucleic acid, the detection domain forms a right-wound double-stranded structure together with one shared base included in the test nucleic acid, and a different base pair is formed depending on whether or not the shared base is a predetermined base. Also provided is a detection method using the nucleobase detection agent.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a nucleic acid base detection agent, a composition, a kit and a device using the same, and a method for detecting a target nucleic acid. [Background technology]

[0002] It is known that single nucleotide polymorphisms are related to phenotypes such as the degree of side effects to drugs and susceptibility to diseases. In plants, single nucleotide polymorphisms are also related to phenotypes such as disease resistance, stress tolerance, and biomass volume. In particular, it has become clear that post-transcriptional modification mutations and SNPs increase the risk of cancer or cause its onset. In this way, by detecting single nucleotide polymorphisms, it is possible to predict the risk of contracting a disease or the risk of developing side effects, and attention is also being paid to personalized medicine, which provides treatments and other treatments that enable optimal treatment and drug administration for individual patients based on this information.

[0003] Known methods for detecting single nucleotide polymorphisms include, for example, a direct sequencing method in which a DNA fragment containing a single nucleotide polymorphism is amplified to read the base sequence, a method using a probe containing a single nucleotide polymorphism site, a method using a protein that exhibits different behavior depending on the presence of a mismatch, etc. Methods using a probe containing a single nucleotide polymorphism site include a cycling probe method that applies real-time PCR, a bead array method, a microarray (DNA chip) method, etc.

[0004] Among these, the method using a probe is particularly useful from the viewpoints of the simplicity of the detection method based on the small number of steps, the ease of design according to the type of SNP, etc., and among them, the method using a molecular beacon is known as a particularly simple and inexpensive method. A typical molecular beacon has a base sequence in its center that is complementary to the wild-type sequence of the target single-stranded DNA or RNA, and has base sequences complementary to each other at both ends. Furthermore, a fluorescent substance and a quencher are bound to each of these ends (Non-Patent Document 1).

[0005] Molecular beacons function as follows. In the absence of a target molecule, the molecular beacon hybridizes at both ends, bringing the fluorophore and quencher in close proximity and not emitting fluorescence. In the presence of a wild-type target sequence, the molecular beacon cleaves the double-stranded region formed at both ends and hybridizes to the target sequence. This separates the fluorophore and quencher, causing fluorescence. On the other hand, if a mutation occurs in the target sequence due to an SNP, it becomes difficult for the molecular beacon to hybridize, and the fluorescence remains quenched. As a result, the presence of an SNP can be detected by the lack of fluorescence. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Tyagi, S., & Kramer, FR (1996) Nature biotechnology, 14(3), 303-308. Summary of the Invention [Problem to be solved by the invention]

[0007] The mechanism of action of molecular beacons is simple, so the number of detection steps and types of reagents required is small, and the molecular beacons themselves are easy to design. On the other hand, because SNPs are minute mutations of a single base, molecular beacons may hybridize weakly with the target sequence even when they contain mismatches, making it difficult to detect SNPs. In addition, when there is a mutation in a base other than the SNP or when the target sequence does not exist, hybridization does not occur and the fluorescence remains quenched regardless of the presence or absence of the SNP, making it difficult to distinguish from cases where an SNP is detected.

[0008] Furthermore, although differences in modification patterns based on post-transcriptional modifications of mRNA have been attracting attention in recent years, no technology was available that could detect them.

[0009] An object of the present invention is to provide a highly sensitive means for detecting mutations which is easy to design and detect, similarly to molecular beacons, and is inexpensive, and which is also capable of detecting differences in modification patterns. [Means for solving the problem]

[0010] In order to solve the above problems, the present inventors have conducted intensive research and found that, rather than relying on the presence or absence of hybridization, local changes in bases can be converted into changes in three-dimensional structure by devising a probe design, making them directly detectable. Furthermore, it has been found that this method also makes it possible to detect differences in modification patterns. The present invention is based on these novel findings and provides the following: [1] A nucleobase detection agent for detecting a specified base in a test nucleic acid, the nucleobase detection agent comprising a nucleic acid molecule including a base sequence region capable of hybridizing to the test nucleic acid, the base sequence region including a detection domain consisting of one working base and one indicator base adjacent to it, the nucleic acid molecule being configured such that, when hybridized to the test nucleic acid, the detection domain forms a right-handed double-stranded structure together with one shared base included in the test nucleic acid, and when the shared base is other than the specified base, a base pair is formed between the shared base and the indicator base, and when the shared base is the specified base, a base pair is formed between the shared base and the working base. [2] The nucleic acid base detection agent according to [1], wherein the base sequence region has a length of 6 bases or more. [3] The nucleic acid base detection agent according to [1] or [2], wherein the nucleic acid molecule comprises a plurality of the detection domains. [4] The nucleic acid base detection agent according to any one of [1] to [3], wherein the predetermined base is one or more bases selected from the group consisting of adenine, thymine, guanine, cytosine, uracil, 6-methyladenine, 4-methylthymine, 6-methylguanine, 7-methylguanine, 5-methylcytosine, and 5-methyluracil. [5] A composition for detecting a specific base, comprising the nucleic acid base detection agent according to any one of [1] to [4]. [6] A composition for detecting a point mutation, comprising the nucleic acid base detector according to any one of [1] to [4]. [7] The composition for detection described in [6], wherein the point mutation is a single nucleotide polymorphism. [8] The composition for detection according to any one of [5] to [7], which contains multiple types of nucleic acid base detection agents. [9] A kit for detecting a specific base, comprising the nucleic acid base detection agent according to any one of [1] to [4] and / or the detection composition according to any one of [5] to [8].

[10] A device for detecting a predetermined base, comprising the nucleic acid base detection agent according to any one of [1] to [4] and / or the detection composition according to any one of [5] to [8].

[11] A method for detecting a target nucleic acid containing a specified base as a covalent base, comprising: a contacting step of contacting a test sample containing a nucleic acid with a nucleic acid base detection agent described in any one of [1] to [4] and / or a detection composition described in any one of [5] to [8]; a detection step of detecting a protrusion of the indicated base from the double-stranded structure; and a determination step of determining that the target nucleic acid is present in the test sample when the protrusion of the indicated base is detected.

[12] The detection method described in

[11] , wherein the test sample is in a form selected from the group consisting of liquid, semisolid, and aerosol. Effect of the Invention

[0011] According to the nucleic acid base detection agent of the present invention, a predetermined base can be detected specifically and with high sensitivity.

[0012] Furthermore, the detection composition of the present invention makes it possible to detect the presence of a predetermined base specifically and with high sensitivity.

[0013] Furthermore, according to the detection method of the present invention, a predetermined base in a test sample can be detected. [Brief description of the drawings]

[0014] [Figure 1]FIG. 1 is a schematic diagram showing an exemplary structure of a nucleic acid molecule of the present invention. FIG. 1A shows the structure of a nucleic acid molecule when the shared base is a base other than a predetermined base. FIG. 1B shows the structure of a nucleic acid molecule when the shared base is a predetermined base. In the figure, the dashed line between two base sequence regions indicates that base pairs are formed. In the figure, the dashed frame indicates an exemplary sensing domain. [Diagram 2] FIG. 2 is a schematic diagram showing the structure of the nucleic acid molecule used in the examples. FIG. 2A shows the structure of the nucleic acid molecule used in Example 1 when hybridized with a test nucleic acid. FIG. 2B shows the structure of the nucleic acid molecule used in Example 2 when hybridized with a test nucleic acid. In the figure, A is adenine, T is thymine, U is uracil, C is cytosine, G is guanine, and 2AP is 2-aminopurine. In FIG. 2A, "X1" refers to a shared base, which is either adenine, uracil, cytosine, or guanine. In FIG. 2A, "Y1" refers to a working base, which is either adenine, thymine, cytosine, or guanine. In FIG. 2B, "X2" refers to a shared base, which is either adenine, 6-methyladenine, cytosine, or 5-methylcytosine. In FIG. 2B, "Y2" refers to a working base, which is thymine or guanine. In the figure, the horizontal black lines indicate that base pairs are formed between nucleic acid strands. [Diagram 3] Fig. 3 shows the change in fluorescence intensity at an excitation wavelength of 305 nm when various RNA molecules with different shared bases are added as test nucleic acids to a solution containing an adenine detection nucleic acid molecule containing thymine as a working base. Fig. 3A shows the results of measuring the fluorescence spectrum of a solution to which various RNA molecules have been added. Fig. 3B shows the rate of change (ΔF) in fluorescence intensity at a wavelength of 375 nm under each condition of Fig. 3A. In the figure, "RNA-A" indicates an RNA molecule containing adenine as a shared base, "RNA-G" indicates an RNA molecule containing guanine as a shared base, "RNA-C" indicates an RNA molecule containing cytosine as a shared base, and "RNA-U" indicates an RNA molecule containing uracil as a shared base. [Figure 4]Fig. 4 shows the change in fluorescence intensity at an excitation wavelength of 305 nm when various RNA molecules with different shared bases are added as test nucleic acids to a solution containing a cytosine detection nucleic acid molecule containing guanine as the working base. Fig. 4A shows the results of measuring the fluorescence spectrum of a solution to which various RNA molecules have been added. Fig. 4B shows the rate of change (ΔF) in fluorescence intensity at a wavelength of 370 nm under each condition of Fig. 4A. In the figure, "RNA-A" indicates an RNA molecule containing adenine as the shared base, "RNA-G" indicates an RNA molecule containing guanine as the shared base, "RNA-C" indicates an RNA molecule containing cytosine as the shared base, and "RNA-U" indicates an RNA molecule containing uracil as the shared base. [Diagram 5] Fig. 5 shows the change in fluorescence intensity at an excitation wavelength of 305 nm when various RNA molecules with different shared bases are added as test nucleic acids to a solution containing a guanine detection nucleic acid molecule containing cytosine as the working base. Fig. 5A shows the results of measuring the fluorescence spectrum of a solution to which various RNA molecules have been added. Fig. 5B shows the rate of change (ΔF) in fluorescence intensity at a wavelength of 370 nm under each condition of Fig. 5A. In the figure, "RNA-A" indicates an RNA molecule containing adenine as a shared base, "RNA-G" indicates an RNA molecule containing guanine as a shared base, "RNA-C" indicates an RNA molecule containing cytosine as a shared base, and "RNA-U" indicates an RNA molecule containing uracil as a shared base. [Figure 6] Fig. 6 shows the change in fluorescence intensity at an excitation wavelength of 305 nm when various RNA molecules with different shared bases are added as test nucleic acids to a solution containing a uracil detection nucleic acid molecule containing adenine as the working base. Fig. 6A shows the results of measuring the fluorescence spectrum of a solution to which various RNA molecules have been added. Fig. 6B shows the rate of change (ΔF) in fluorescence intensity at a wavelength of 360 nm under each condition of Fig. 6A. In the figure, "RNA-A" indicates an RNA molecule containing adenine as the shared base, "RNA-G" indicates an RNA molecule containing guanine as the shared base, "RNA-C" indicates an RNA molecule containing cytosine as the shared base, and "RNA-U" indicates an RNA molecule containing uracil as the shared base. [Figure 7]Fig. 7 shows the change in fluorescence intensity at an excitation wavelength of 305 nm when various RNA molecules with different shared bases are added as test nucleic acids to a solution containing an adenine detection nucleic acid molecule containing thymine as the working base. Fig. 7A shows the results of measuring the fluorescence spectrum of a solution to which various RNA molecules were added. Fig. 7B shows the rate of change (ΔF) in fluorescence intensity at a wavelength of 375 nm under each condition of Fig. 7A. In the figure, "RNA-A" shows an RNA molecule containing adenine as the shared base, and "RNA-m6A" shows an RNA molecule containing 6-methyladenine as the shared base. [Figure 8] Fig. 8 shows the change in fluorescence intensity at an excitation wavelength of 305 nm when various RNA molecules with different shared bases are added as test nucleic acids to a solution containing a cytosine detection nucleic acid molecule containing guanine as the working base. Fig. 8A shows the results of measuring the fluorescence spectrum of a solution to which various RNA molecules were added. Fig. 8B shows the rate of change (ΔF) in fluorescence intensity at a wavelength of 370 nm under each condition of Fig. 8A. In the figure, "RNA-C" shows an RNA molecule containing cytosine as the shared base, and "RNA-m5C" shows an RNA molecule containing 5-methylcytosine as the shared base. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] 1. Nucleic acid base detection agent 1-1. Overview The first aspect of the present invention is a nucleic acid base detection agent for detecting a predetermined base in a test nucleic acid. The nucleic acid base detection agent of the present invention includes a nucleic acid molecule containing two base sequence regions that can hybridize with each other as an essential component, and detects a predetermined base. The nucleic acid base detection agent of the present invention can be an active ingredient of the detection composition, detection kit and detection device of the present invention described later.

[0016] 1-2.Definition Terms used in this specification are defined below. The term "nucleic acid" or "nucleic acid molecule" refers to a biopolymer in which nucleotides are structural units linked together by phosphodiester bonds. Nucleic acids can be broadly classified into natural nucleic acids and artificial nucleic acids, but both are included in the present specification.

[0017] "Natural nucleic acid" refers to a nucleic acid that exists in nature. For example, this includes DNA and RNA. "Artificial nucleic acid" refers to a nucleic acid molecule that is artificially synthesized by biological methods or chemical synthesis methods. Unless otherwise specified, the artificial nucleic acid in this specification may be composed of, for example, all unmodified natural nucleotides, or may contain non-natural nucleotides or modified nucleotides.

[0018] "Nucleotide" refers to a molecule in which a phosphate group is covalently linked to the sugar portion of a nucleoside. In the case of nucleotides that contain a pentofuranosyl sugar, the phosphate group is usually linked to the 3' or 5' hydroxyl group of the sugar.

[0019] "Nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar is usually, but not limited to, a pentofuranosyl sugar. Examples of pentofuranosyl sugars include ribose and deoxyribose. Examples of bases (nucleobases) include adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). In this specification, unless otherwise specified, the base may be either a modified base or an unmodified base.

[0020] As used herein, the term "modification" refers to partial or complete substitution of a nucleotide, which is a structural unit of a nucleic acid, or a nucleoside, which is a structural component thereof, with another atomic group, or addition of a functional group, etc. Specific examples of such modification include sugar modification, base modification, and modification of a phosphodiester bond.

[0021] As used herein, the term "modified nucleotide" refers to a nucleotide in which a part or all of a nucleotide has been replaced with another atomic group or to which a functional group or the like has been added. The term "unmodified nucleotide" refers to a nucleotide other than modified nucleotides. In principle, most naturally occurring nucleotides fall into this category.

[0022] As used herein, "sugar modification" refers to a substitution and / or any change in the sugar moiety of a nucleic acid molecule.

[0023] "Modified base" refers to a nucleobase other than the naturally occurring adenine, cytosine, guanine, thymine, or uracil, and "base modification" refers to a change to such a nucleobase.

[0024] An "artificial base" refers to an artificially constructed chemical substance that has properties similar to those of a natural base and is capable of forming a specific base pair. As used herein, "capable of forming a specific base pair" refers to being capable of forming a base pair with one or more types of natural bases and / or one or more types of artificial bases.

[0025] The term "base sequence" refers to the order of nucleotides covalently bonded in a nucleic acid molecule, expressed by the bases of each nucleotide. In addition, the term "base sequence region" as used herein refers to a continuous nucleic acid region that constitutes a nucleic acid chain and has a specific base sequence.

[0026] "Right-handed double-stranded structure" refers to a double-stranded structure formed by a nucleic acid molecule that has a clockwise helical structure. Specifically, this includes A-type and B-type structures. "A-type structure" refers to a right-handed double-stranded structure with 11 bases per turn and an average helical diameter of 20 Å. In general, double-stranded RNA and RNA-DNA double-stranded structures tend to have A-type structures. On the other hand, "B-type structure" refers to a right-handed double-stranded structure with 10 bases per turn and an average helical diameter of 23 Å. In general, double-stranded DNA tends to have B-type structures.

[0027] The term "base pair" refers to a pairing of two bases that constitute a nucleic acid molecule. In a base pair consisting of two bases, the bases are usually paired by hydrogen bonds.

[0028] In this specification, a base pair of base A and base B is represented as "A / B". In any of these cases, the order is not limited to the order described. For example, unless otherwise specified, either base A or base B can be a shared base. Meanwhile, in this specification, base A and base B are bases of nucleotides adjacent to each other on the same nucleic acid chain, and are represented as "AB".

[0029] As used herein, the phrase "capable of forming a base pair" with a specific base refers to a base having a structure capable of forming a base pair with the specific base and being in a positional relationship capable of forming a base pair with the specific base.

[0030] In this specification, the phrase "a specific base pair is formed" for a certain base means that the specific base pair is energetically stable compared to other base pairs that the base can form. With respect to a bond, "more energetically stable" means that the bond energy of the bond is greater than that of the comparison target. "Bond energy" refers to the energy required to break the bond between 1 mol of atoms.

[0031] "Detection" refers to testing to find the presence of a particular component or a particular state. In particular, in this specification, it refers to finding the presence of a predetermined base in a test sample.

[0032] "Point mutation" refers to a mutation that causes modification, substitution, insertion, or deletion of a single nucleotide base of genetic material, DNA, or RNA. Point mutations based on modification include methylation, deamination, thiolation, etc., and any of these mutations may be used. Point mutations based on substitution include transition mutations, which are substitutions between purines or pyrimidines, and transversion mutations, which are substitutions between purines and pyrimidines, and any of these mutations may be used. In addition, point mutations in this specification include both congenital and acquired mutations. Furthermore, point mutations include, but are not limited to, missense mutations that result in amino acid substitutions, nonsense mutations that result in stop codons, silent mutations that do not result in amino acid substitutions due to substitution with degenerate codons, and splice site mutations.

[0033] "Genetic polymorphism" refers to a mutation in genomic DNA that is held by 1% or more of individuals of the same species. "Single nucleotide polymorphism", also called "SNP", refers to a genetic polymorphism in which the base sequence on genomic DNA differs by only one base. In this specification, the single nucleotide polymorphism broadly includes a mutation in which the base sequence on genomic DNA differs by only one base even if the frequency is less than 1%. It is believed that there are a total of about 10 million SNPs throughout the genome. Genome SNP information can be obtained from databases such as dbSNP (https: / / www.ncbi.nlm.nih.gov / snp / ) of the National Center for Biotechnology Information (NCBI). In particular, hundreds of thousands to millions of SNPs are said to be highly related to human diseases, biological reactions, drug sensitivity, etc. Single nucleotide polymorphisms held by one individual on genomic DNA typically consist of two types, a wild type allele and one type of mutant allele, but there are also cases where there are two or more types of mutant alleles. "Contact" means direct physical contact of one substance with another substance.

[0034] 1-3.Configuration The nucleic acid base detection agent of the present invention includes, as an essential component, a nucleic acid molecule including a base sequence region capable of hybridizing to a test nucleic acid. The nucleic acid molecule also includes a detection domain that forms a right-handed double-stranded structure together with one shared base contained in the test nucleic acid when hybridized with the test nucleic acid. Each component will be described in detail below.

[0035] 1-3-1. Base sequence region The nucleic acid molecule contained in the nucleic acid base detector of the present invention contains a base sequence region and is capable of hybridizing to a test nucleic acid.

[0036] As used herein, the term "hybridize" refers to the formation of a double strand by polynucleotides having completely or partially complementary base sequences.

[0037] The length of the hybridizing base sequence region is not particularly limited. For example, it may be 6 bases to 120 bases. Specifically, for example, it may be 6 bases or more, 7 bases or more, 8 bases or more, 9 bases or more, 10 bases or more, 11 bases or more, 12 bases or more, 13 bases or more, 14 bases or more, 15 bases or more, 16 bases or more, 17 bases or more, 18 bases or more, 19 bases or more, 20 bases or more, 21 bases or more, 22 bases or more, or, for example, 120 bases or less, 110 bases or less, 100 bases or less, 90 bases or less, 80 bases or less, 70 bases or less, 60 bases or less, 50 bases or less, 40 bases or less, or 30 bases or less. The base sequence of this base sequence region is not particularly limited as long as it can hybridize to the test nucleic acid.

[0038] The hybridization conditions are not particularly limited, and may be various stringent conditions, such as low stringent conditions and high stringent conditions. Preferably, hybridization may be performed under high stringent conditions. "High stringent conditions" refers to conditions under which non-specific hybrids are not formed. The low salt concentration referred to here specifically refers to, for example, 15 to 750 mM, preferably 15 to 500 mM, 15 to 300 mM, or 15 to 200 mM. The high temperature referred to here specifically refers to, for example, 50 to 68°C, or 55 to 70°C. Specific examples of high stringent conditions include conditions at 65°C, washing with 0.1xSSC and 0.1% SDS. Here, 1xSSC contains 150 mM sodium chloride and 15 mM sodium citrate.

[0039] Whether or not a nucleic acid can hybridize can be determined using a method known in the art. For example, it can be determined based on base identity. Usually, a second nucleic acid having a base sequence that is completely complementary to the base sequence of a first nucleic acid or a base sequence with a certain level of base identity can hybridize with the first nucleic acid. Specifically, for example, a second nucleic acid can hybridize when the base identity is 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0040] As used herein, "base identity" refers to the percentage (%) of identical bases in one polynucleotide relative to the total number of bases in the other polynucleotide when the base sequences of two polynucleotides are aligned and, if necessary, gaps are introduced into one of the base sequences to maximize the degree of base identity between the two. The % identity can be easily determined using a known program such as the homology search program BLAST (Basic local alignment search tool; Altschul, SF et al., J. Mol. Biol., 215, 403-410, 1990) search. In addition, a second nucleic acid having a base sequence in which one or more bases are replaced with other bases in a sequence completely complementary to the base sequence of a first nucleic acid can usually hybridize with the first nucleic acid.

[0041] In this specification, "plurality" refers to two or more, and specifically refers to, for example, 2 to 30, 2 to 14, 2 to 12, 2 to 10, for example, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 (several). For example, one or more base pairs adjacent to both ends or one end of the detection domain can be complementary base pairs.

[0042] The specific base sequence of the base sequence region is not particularly limited as long as the detection domain forms a right-handed double-stranded structure together with the shared base of the test nucleic acid when hybridized with the test nucleic acid. Preferably, the sequence on the 5'-end and / or 3'-end of the detection domain is a sequence known to form a right-handed double-stranded structure.

[0043] For example, a right-handed double-stranded structure can be obtained by avoiding sequences known to form left-handed double-stranded structures. Specific sequences known to form left-handed double-stranded structures include, for example, GC repeat sequences (specifically, for example, (GC) n (n is a natural number of 2 or more) and other repeating sequences are known.

[0044] The length of the sequence that forms a right-handed double-stranded structure is not particularly limited, but for example, the sequence on the 3'-end and / or 5'-end of the detection domain can be 1 base or more, 2 bases or more, 3 bases or more, 4 bases or more, 5 bases or more, 6 bases or more, 7 bases or more, 8 bases or more, 9 bases or more, or 10 bases or more.

[0045] Whether or not a target sequence can form a right-handed double-stranded structure can be known using a method known in the art. For example, when a sequence motif or consensus sequence known to form a right-handed double-stranded structure is used, it can be presumed that the specific three-dimensional structure is formed. In this case, it is not necessary to confirm whether or not a right-handed double-stranded structure is actually formed. On the other hand, when confirming that a right-handed double-stranded structure is actually formed, it can be confirmed, for example, by in silico analysis, structural analysis, or by observing a phenomenon that occurs specifically in a right-handed double-stranded structure.

[0046] For in silico analysis, a three-dimensional structure prediction program known in the art, such as RNAComposer, RNAMotifScan, 3dRNA, ModeRNA, MacroMoleculeBuilder, NAST, iFoldRNA, Vfold3D, SimRNA, or a combination thereof, can be used.

[0047] When confirmation is performed by structural analysis, the conditions and methods used for crystallization and structural analysis are not particularly limited. Specific methods include, for example, circular dichroism spectrum (CD spectrum) analysis, neutron crystallography, nuclear magnetic resonance (NMR), X-ray crystal structure analysis, melting temperature measurement of a nucleic acid double strand, or a combination thereof.

[0048] Alternatively, the formation of a three-dimensional structure can be confirmed by observing a phenomenon that occurs specifically in a right-handed double-stranded structure. For example, proteins such as ZBP1 and ADAR1 are known to bind to DNA specifically in a left-handed Z-shaped structure. Therefore, by examining the binding of these proteins, it can be confirmed whether the target sequence forms a right-handed structure.

[0049] 1-3-2. Detection domain The nucleic acid molecule contained in the nucleic acid base detector of the present invention contains one or more sensing domains, and the sensing domain consists of one working base and one indicator base (FIG. 1).

[0050] In the sensing domain, the driver and indicator bases are adjacent to each other (FIG. 1) and can interact with a shared base when hybridized to a test nucleic acid.

[0051] The shared base is contained in the test nucleic acid (Figure 1) and is capable of forming a base pair with the indicator base (Figure 1A) and, if the shared base is a predetermined base, is capable of forming a base pair with the worker base (Figure 1B).

[0052] A sensing domain in the present invention behaves as follows. When the nucleic acid molecule of the present invention hybridizes with a test nucleic acid, if the shared base in the test nucleic acid is other than the specified base (FIG. 1A), a base pair is formed between the shared base and the indicated base. At this time, the working base that cannot participate in the base pairing protrudes outside the double-stranded structure.

[0053] On the other hand, when the shared base in the test nucleic acid is a specific base (Figure 1B), a base pair is formed between the shared base and the working base, and the indicator base that cannot participate in the base pairing protrudes outside the double-stranded structure.

[0054] In this way, the detection domain changes its three-dimensional structure depending on whether the shared base is a specific base or not, and as a result, the base protruding outward from the double-stranded structure changes, making it possible to observe whether the detection domain is in contact with the specific base from the outside of the nucleic acid molecule.

[0055] (1) Working base The working base is a base that protrudes outside the double-stranded structure when the shared base is other than the predetermined base, and is capable of forming a base pair with the shared base when the shared base is the predetermined base. The working base is not particularly limited as long as it satisfies the relationship between bases described below.

[0056] The working base may be a natural base or an artificial base, and may be a modified or unmodified base.

[0057] Usually, according to the nucleic acid base detection agent of the present invention, the overhang of the indicator base is detected to detect that the common base of the test nucleic acid is a predetermined base, so there is no need to detect the overhang of the actuator base. However, the actuator base may be detectable. In this case, for example, the overhang of the indicator base may be detected when the overhang of the actuator base is not detected. Also, for example, both the overhang of the actuator base and the overhang of the indicator base may be detected.

[0058] In this case, the detectable base that can be used as the working base may be, for example, any of the various bases described below as indicator bases.

[0059] When both the working base and the indicator base are detectable, it is preferred that each is separately detectable.

[0060] As used herein, the term "separately detectable" refers to being detectable separately from one another, including the case where they are detectable by using the same method but different conditions, such as fluorescence of different wavelengths, and the case where they are detectable by different methods, such as color development and fluorescence.

[0061] (2) Display base The indicator base is capable of forming a base pair with the shared base when the shared base is other than the predetermined base, and is a base that protrudes outward from the double-stranded structure when the shared base is the predetermined base. The indicator base is not particularly limited as long as it satisfies the relationship between bases described below.

[0062] The nucleic acid base detector of the present invention detects that the shared base of the test nucleic acid is a predetermined base by detecting the protrusion of the indicator base from the double-stranded structure. Therefore, it is preferable that the indicator base is a base that can detect the protrusion.

[0063] For example, when the indicated base is a natural base, it can be detected using an antibody, antigen-binding fragment, aptamer, etc. capable of specifically binding to the indicated base.

[0064] The indicator base may also contain a detectable label. The type of detectable label is not particularly limited and may be appropriately determined depending on the detection method. Specific detectable labels include, for example, fluorescent dyes (e.g., FITC, Texas, Cy3, Cy5, Cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493, NBD, and TAMRA), luminescent substances (e.g., acridinium esters), non-colored small molecules that act as enzyme substrates or antigens (e.g., biotin and DIG), and radioisotopes (e.g., 32 P, 3 H and 14 The label used for the indicator base is preferably a label that can be detected by distinguishing between a case where the indicator base protrudes outside the double-stranded structure and a case where it does not protrude outside the double-stranded structure.

[0065] In addition, a fluorescent base can also be used as a label. The type of fluorescent base is not particularly limited as long as it is a nucleic acid base that emits fluorescence. Specific fluorescent bases are described, for example, in the Glen research catalog (https: / / www.glenresearch.com / media / / folio3 / productattachments / product_catalog / Glen_Product_Catalog_2021.pdf), Bood, M., et al., Beilstein Journal of Organic Chemistry, 2018, 14(1), 114-129., etc. Fluorescent bases include, but are not limited to, 2-aminopurine, pA, qA and their derivatives, 1,N 6-ethenoadenine, etc.; cytosine analogues such as pyrrolocytosine, tC(1,3-diaza-2-oxophenoxazine), 9-aminoethyl-1,3-diaza-2-oxophenoxazine, 1,3-diaza-2-oxophenothiazine, and derivatives thereof; uracil analogues such as 5-(1-pyrenyl-ethynyl)uracil, etc. The fluorescent base may be attached, for example, to the 1' position of the sugar.

[0066] The nucleotide having a fluorescent base may be, for example, a deoxyribonucleotide having a 2-aminopurine having the following structure:

[0067] [ka]

[0068] The nucleotide having a fluorescent base may be, for example, a deoxyribonucleotide having pA having the following structure or a derivative thereof, where R is deoxyribose:

[0069] [ka]

[0070] The nucleotide having a fluorescent base may be, for example, a deoxyribonucleotide having the following structure: qA, or a derivative thereof, where R is deoxyribose.

[0071] [ka]

[0072] The nucleotide having a fluorescent base may also be a deoxyribonucleotide having, for example, a pyrrolocytosine having the following structure:

[0073] [ka]

[0074] (3) Relationship between bases The position of the shared base relative to the indicated base is not particularly limited as long as it can form a base pair. For example, when the indicated base in the base sequence region is the Mth base from the 5' end of the base sequence region, the shared base can be the M-1th, Mth, or M+1th base from the 3' end of the nucleic acid region that hybridizes with the base sequence region of the test nucleic acid.

[0075] In addition, the position of the shared base relative to the working base is not particularly limited as long as it can form a base pair. For example, when the working base in the base sequence region is the Nth base from the 5' end of the base sequence region, the shared base can be the N-1th, Nth, or N+1th base from the 3' end of the nucleic acid region that hybridizes with the base sequence region of the test nucleic acid.

[0076] Furthermore, the position of the actuator base relative to the display base is not particularly limited as long as it is adjacent. For example, when the position of the display base in the base sequence region is Mth from the 5' end, the position of the actuator base (Nth from the 5' end) can be M-1th or M+1th from the 5' end of the base sequence region. Preferably, when the position of the display base is Mth from the 5' end of the base sequence region, the position of the actuator base (Nth from the 5' end) is M+1th from the 5' end of the base sequence region.

[0077] Preferably, when the indicator base and the working base in one base sequence region are located at the Mth and M+1th positions from the 5' end, respectively, the shared base is located at the Mth position from the 3' end of the nucleic acid region that hybridizes with the base sequence region of the test nucleic acid.

[0078] As described above, the sensing domain changes its shape depending on whether the shared base is a predetermined base, as follows: If the shared base in the test nucleic acid is other than the designated base, a base pair is formed between the shared base and the designated base, and the working base protrudes outside the double-stranded structure; When the shared base in the test nucleic acid is a predetermined base, a base pair is formed between the shared base and the worker base, and the indicator base protrudes outside the double-stranded structure.

[0079] The types of the indicator base and the actuator base are not particularly limited. For example, the indicator base and the actuator base may be the same or may be bases having the same energetic stability of the base pair bond with the predetermined base, or may be different types or bases having different energetic stability of the base pair bond with the predetermined base.

[0080] When the shared base is other than the specified base, it is preferable that the bond between the shared base and the indicator base is more energetically stable than the base pair between the shared base and the worker base, or the bond stability is equivalent. When the indicator base and the worker base are arranged in this order from the 5'-end, if the bond stability of the base pair with the shared base of both bases is equivalent, the shared base other than the specified base and the indicator base usually form a base pair.

[0081] When the shared base is a predetermined base, it is preferable that the bond between the shared base and the working base is more energetically stable than the base pair between the shared base and the indicator base, or the bond stability is equivalent. When the indicator base and the working base are arranged in this order from the 5'-end, if the bond stability of the base pair with the shared base of both bases is equivalent, the shared base, which is a predetermined base, and the working base usually form a base pair.

[0082] As used herein, "having equivalent binding stability" with respect to two types of base pairs means that there is no significant difference in the frequency of formation of the first base pair and the second base pair under conditions in which the first base pair and the second base pair can be formed independently.

[0083] The energetic stability of a particular base pair can be estimated using techniques and information known in the art (see, for example, Sherer, EC, et al., (2003) Journal of computational chemistry, 24(1), 57-67.; Takezawa, Y., et al., (2017) Chemistry Letters, 46(5), 622-633.). Specifically, for example, PM3 BP The energy stability of the base pair of the sensing domain when the shared base is a predetermined base and when the shared base is other than the predetermined base can be experimentally confirmed by using a technique such as measuring the melting temperature of a nucleic acid double strand.

[0084] The base pairs of the shared base and the driver base and the base pairs of the shared base and the indicator base may be either complementary or non-complementary base pairs. Preferably, the base pairs of the shared base and the driver base are complementary base pairs.

[0085] In addition, the relationship of energy stability may be established under the conditions of using the specific nucleic acid base detector of the present invention.For example, under the conditions of specific pH, temperature, metal ion concentration, etc., when the above-mentioned relationship is established, the nucleic acid base detector having the detection domain containing these base pairs can be used under those conditions.

[0086] The base structure of the detection domain used in the present invention is not particularly limited as long as it satisfies the above-mentioned requirements. Specifically, for example, A(m 6 A) / T(U,m 4 T)-A(m 6 A), A(m 6 A) / T(U,m 4 T)-(2-aminopurine), T(U,m 4 T) / A(m 6 A)-A(m 6A) (e.g. U / AA), T(U, m 4 T) / A(m 6 A)-(2-aminopurine) (e.g. U / A-(2-aminopurine) etc.), C(m 5 C) / G(m 7 G, m 6 G)-A(m 6 A), C(m 5 C) / G(m 7 G, m 6 G)-(2-aminopurine), G(m 7 G, m 6 G) / C(m 5 C)-A(m 6 A), G(m 7 G, m 6 G) / C(m 5 C)-(2-aminopurine), A(m 6 A) / T(U,m 4 T)-(1,N 6 -ethenoadenine), T(U,m 4 T) / A(m 6 A)-(1,N 6 -ethenoadenine), C(m 5 C) / G(m 7 G, m 6 G)-(1,N 6 -ethenoadenine), G(m 7 G, m 6 G) / C(m 5 C)-(1,N 6 -ethenoadenine), A(m 6 A) / T(U,m 4 T)-C(m 5 C), A(m 6 A) / T(U,m 4 T)-(pyrrolocytosine), T(U,m 4 T) / A(m 6 A)-C(m 5 C) (e.g. U / AC, etc.), T(U, m 4 T) / A(m 6 A)-(pyrrolocytosine) (e.g. U / A-(pyrrolocytosine) etc.), C(m 5 C) / G(m 7 G, m 6 G)-C(m 5 C), C(m 5 C) / G(m7 G, m 6 G)-(pyrrolocytosine), G(m 7 G, m 6 G) / C(m 5 C)-C(m 5 C), G(m 7 G, m 6 G) / C(m 5 Here, the description "base 1 (base 2)" indicates that base 2 can be used in place of base 1. However, even if the base 2 is not one of the bases exemplified here, it can be used as long as it is a derivative or analog of the same base as base 1. In addition, 2-aminopurine and 1,N 6 Instead of -ethenoadenine, other adenine analogs exemplified for the indicator bases can be used, and instead of pyrrolocytosine, other cytosine analogs exemplified for the indicator bases can be used, etc. Furthermore, when the predetermined base and the working base are complementary, the type of indicator base is not particularly limited, and any base (including a fluorescent base) can be used as the indicator base.

[0087] The detection domain forms a right-handed double-stranded structure. The right-handed double-stranded structure in this specification may be either type A or type B, but is preferably type A. Usually, when the sequence on the 5'-end and / or 3'-end side of the detection domain forms a right-handed double-stranded structure, the detection domain also forms a right-handed double-stranded structure.

[0088] In addition, when the nucleic acid molecule or polymer thereof of the present invention contains multiple detection domains, the positional relationship between them is not particularly limited. For example, the display bases of different detection domains can be positioned so that they are not sterically hindered from each other. Specifically, for example, the display bases may be separated by 3 or more base pairs or 4 or more base pairs.

[0089] 1-3-3. Nucleic acid molecules The base sequence of the nucleic acid molecule of the present invention is not particularly limited. The nucleic acid molecule of the present invention can include one or more of the above-mentioned base sequence regions. When including multiple base sequence regions, the number of bases between the regions is not particularly limited. For example, it can be 1 base or more, 2 bases or more, 3 bases or more, 4 bases or more, 5 bases or more, 6 bases or more, 7 bases or more, 8 bases or more, 9 bases or more, 10 bases or more, 20 bases or more, 50 bases or more, 100 bases or more, etc.

[0090] The nucleic acid molecule of the present invention can contain one or more detection domains. When the nucleic acid molecule contains multiple detection domains, it may contain multiple detection domains of the same type or different types of detection domains. In addition, for example, when it contains two or more types of detection domains, it is possible to use detection domains that detect the same or different predetermined bases. Specifically, by containing multiple detection domains that detect the same predetermined base, it is possible to increase the detection efficiency. In addition, by containing multiple detection domains that detect different predetermined bases, it is possible to detect multiple types of predetermined bases with one nucleic acid base detection agent.

[0091] The nucleic acid molecule may contain an additional base sequence in addition to the base sequence region. The number of bases constituting the additional base sequence is not particularly limited. For example, it may be 1 base or more, 2 bases or more, 3 bases or more, 4 bases or more, 5 bases or more, 6 bases or more, 7 bases or more, 8 bases or more, 9 bases or more, 10 bases or more, 15 bases or more, 20 bases or more, 30 bases or more, 40 bases or more, 50 bases or more, 60 bases or more, or 70 bases or more. It may also be 100 bases or less, 90 bases or less, 80 bases or less, 70 bases or less, 60 bases or less, 50 bases or less, 40 bases or less, 30 bases or less, 20 bases or less, 10 bases or less, 9 bases or less, 8 bases or less, 7 bases or less, 6 bases or less, 5 bases or less, 4 bases or less, 3 bases or less, 2 bases or less, or 1 base.

[0092] The relationship between the additional base sequence and the base sequence region is not particularly limited. For example, a part of the additional base sequence may be hybridizable with a part of the base sequence region, or the additional base sequence may not be hybridizable with the base sequence region.

[0093] The purpose of including an additional base sequence is not particularly limited. For example, the additional base sequence may be included to reduce a signal from a nucleic acid molecule that is not hybridized to a test nucleic acid during detection, to increase the stability of the nucleic acid molecule, or to prevent non-specific binding of the nucleic acid molecule.

[0094] The additional base sequence may be contained within the nucleic acid molecule of the present invention, but may also be contained in the nucleic acid base detection agent as a molecule separate from the nucleic acid molecule of the present invention.

[0095] The type of nucleic acid constituting the nucleic acid molecule of the present invention is not particularly limited. For example, the nucleic acid molecule of the present invention may be composed of DNA, RNA, or a combination thereof, and may contain modified nucleotides.

[0096] Modified nucleotides include both artificially constructed modified nucleotides and naturally occurring modified nucleotides. They include artificial nucleotides (nucleotide analogs) having similar properties and / or structures to unmodified nucleotides, and artificial nucleotides containing modified nucleosides or modified bases having similar properties and / or structures to unmodified nucleosides or unmodified bases that are components of unmodified nucleotides. Specific examples of modified nucleosides include abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, α-deoxyribonucleosides, and β-L-deoxyribonucleosides. Specific examples of modified bases include a 2-oxo(1H)-pyridin-3-yl group, a 5-substituted 2-oxo(1H)-pyridin-3-yl group, a 2-amino-6-(2-thiazolyl)purin-9-yl group, a 2-amino-6-(2-thiazolyl)purin-9-yl group, and a 2-amino-6-(2-oxazolyl)purin-9-yl group.

[0097] The modified nucleotide may include a sugar modification. Specific examples of the modified nucleotide include 2'-O-methylribose (2'-OMe) in which the hydroxyl group at the 2' position is replaced with a methoxy group, 2'-O-ethylribose in which the hydroxyl group at the 2' position is replaced with an ethoxy group, 2'-O-propylribose in which the hydroxyl group is replaced with a propoxy group, or 2'-O-butylribose in which the hydroxyl group is replaced with a butoxy group, 2'-deoxy-2'-fluororibose in which the hydroxyl group is replaced with a fluoro group, or 2'-O-methoxyethylribose (2'-MOE) in which the hydroxyl group is replaced with a 2'-O-methoxyethyl group. The hydroxyl group may be replaced with a functional group other than a hydrocarbon. Specific examples of the modified nucleotide include replacement with H and halogen elements. In addition, the (deoxy)ribose portion of the nucleoside may be replaced with other molecules, such as sugars, morpholino rings, PNA, and XNA. Specifically, examples of such ribose derivatives include arabinose, 2'-fluoro-β-D-arabinose, ribose derivatives in which the hydroxyl group at the 2' position of ribose is bridged with the carbon atom at the 4' position of ribose, and ribose derivatives in which the oxygen at the 4' position of the ribose ring is replaced with sulfur. Also included are those in which the oxygen atom on the ribofuranose ring (the oxygen atom at the 4' position of ribose) is replaced with sulfur. In particular, nucleotides having a bridged ribose derivative are called bridged nucleic acids, and examples thereof include 2'-OMe RNA, 2'-MOE RNA, LNA, 2'-O,5'-NBNA, and 2'-deoxy-trans-3',4'-BNA.

[0098] Modified nucleotides may include modified bases. Examples of modified bases include, but are not limited to, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine or N4-methylcytosine; N6-methyladenine or 8-bromoadenine; 2-thio-thymine; N2-methylguanine or 8-bromoguanine; and 5-methyluracil, 5-fluorouracil, 5-bromouracil, 5-iodouracil or 5-hydroxyuracil.

[0099] For example, modified nucleotides may include naturally occurring modified bases present in non-coding RNA including mRNA, tRNA, rRNA, etc. Examples of such modified bases include inosine, 1-methyladenine, 2-thiothymine, 4-thiothymine, pseudouracil, 2-thiouracil, 4-thiouracil, 5-methoxycarbonylmethyluracil, 5-methoxycarbonylmethyl-2-thiouracil, 2-methylguanine, 8-oxoguanine, 3-methylguanine, 6-thioguanine, 2-dimethylcytosine, 3-methylcytosine, 2-thiocytosine, 4-acetylcytosine, 5-formylcytosine, and the like.

[0100] Each base constituting the nucleic acid molecule of the present invention may be a natural base or an artificial base. For example, adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C) or a derivative thereof, or an artificial ligand may be used. Adenine, thymine, uracil, guanine, cytosine, or a derivative thereof is preferable.

[0101] Examples of adenine derivatives include N1-methyladenine, N6-methyladenine, N6-acetyl-N6-methyladenine, N6-carboxymethyladenine, 2-aminoadenine, 8-aminoadenine, 8-oxoadenine, 8-bromoadenine, 3-deazaadenine, 7-deazaadenine, 1,3-dideazaadenine, 7-deaza-8-azaadenine, ethenoadenine, and phenoxyacetyladenine.

[0102] Examples of thymine and uracil derivatives include 2-thiothymine, 4-thiothymine, O4-methylthymine, O4-carboxymethylthymine, 5,6-dihydrothymine, pseudouracil, 5-bromouracil, 5-fluorouracil, 5-iodouracil, 5-hydroxyuracil, 5-hydroxymethyluracil, 5-methyluracil, 5-methoxycarbonylmethyluracil, 5-methoxycarbonylmethyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5,6-dihydrouracil, O4-triazolyluracil, and the like.

[0103] Examples of guanine derivatives include hypoxanthine, 6-thioguanine, 8-aminoguanine, 8-oxoguanine, 8-bromoguanine, 8-deuteroguanine, 3-methylguanine, O6-methylguanine, O6-carboxymethylguanine, 7-deazaguanine, and 7-deaza-8-azaguanine.

[0104] Examples of cytosine derivatives include 2-thiocytosine, N4-acetylcytosine, N4-ethylcytosine, N4-methylcytosine, N4-carboxymethylcytosine, 5-bromocytosine, 5-iodocytosine, 5-carboxycytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 3-methylcytosine, 5-methylcytosine, 2-dimethylcytosine, 5-methylisocytosine, 5-aza-5,6-dihydrocytosine, pyrrolocytosine, and imidazolecytosine.

[0105] 1-3-4.Test nucleic acid The type of test nucleic acid to be detected by the nucleobase detection agent of the present invention is not particularly limited. The test nucleic acid may be DNA or RNA, and may be an artificial nucleic acid or a natural nucleic acid.

[0106] When the test nucleic acid is a natural nucleic acid, the DNA includes genomic DNA, fragments thereof such as cfDNA, and mixtures thereof, etc. When the test nucleic acid is a natural nucleic acid, the RNA includes non-coding RNA such as rRNA, miRNA, siRNA, snoRNA, snRNA, and mixtures thereof, in addition to mRNA.

[0107] The test nucleic acid contains a shared base, and when the shared base is a predetermined base, it is detected by the nucleic acid base detection agent of the present invention. The types of the shared base and the predetermined base are not particularly limited.

[0108] For example, modified bases can be used as the predetermined base. Specific unmodified bases include adenine, thymine, guanine, cytosine, uracil, and the like.

[0109] The sensing domain is used to detect the presence of a specific base by distinguishing it from other bases. For example, in the case of a nucleic acid base detector in which the specific base to be detected is adenine, the presence of adenine can be detected when the shared base is adenine because the bond between the shared base and the working base is more stable when the shared base is adenine compared to when the shared base is thymine, guanine, cytosine, or uracil.

[0110] For example, a modified base can be used as the predetermined base. The type of modified base is not particularly limited, but examples include bases modified with a hydrocarbon group. For example, 1-methyladenine (m 1 A), 6-methyladenine (m 6 A), 4-methylthymine (m 4 T), 6-methylguanine (m 6 G), 7-methylguanine (m 7 G), 2,2,7-trimethylguanine (m 2,2,7 G), 5-methylcytosine (m 5 C), 5-methyluracil (m 5 In addition to methylated bases such as uracil, isopropyl uracil, inosine, etc., examples of the modified base to be detected in the present specification include one or more bases selected from the group consisting of 6-methyladenine, 4-methylthymine, 6-methylguanine, 7-methylguanine, and 5-methylcytosine.

[0111] Generally, the stability of the base pair bond between the shared base and the working base changes when the shared base is a modified base compared to when the shared base is an unmodified base. Therefore, the presence of an unmodified base can be detected separately from a modified base, or the presence of a modified base can be detected separately from an unmodified base. For example, in the case of a base modified with a hydrocarbon group such as methylation, the bond between the shared base and the working base is more stable when the shared base is a modified base compared to when the shared base is an unmodified base.

[0112] Depending on the change in the binding stability of the base pair between the shared base and the working base, a single detection domain can be used to detect multiple types of predetermined bases.For example, as described above, when the predetermined base is an unmodified base, it can be detected in a manner that distinguishes it from the case where the predetermined base is a modified base.In addition, for example, when the stability of the base pair changes stepwise depending on the type of the shared base, the degree of protrusion of the detected indicator base can be used to determine the type of the shared base.

[0113] 1-3-5.Double-stranded structure When the nucleic acid molecule of the present invention hybridizes with a test nucleic acid, a double-stranded structure is formed. The specific structure of the double-stranded structure is not particularly limited as long as the detection domain is included in the right-handed double-stranded structure. For example, the double-stranded structure may be a linear double-stranded structure as a whole, or may be a double-stranded structure containing branches. The right-handed double-stranded structure may be formed locally or globally.

[0114] The whole region forming the right-handed structure may be composed of either one of A-type or B-type, or may be composed of a combination of A-type and B-type. Preferably, the right-handed structure includes an A-type structure, and for example, at least one of the A-type structures includes a detection domain. When a part of the double-stranded structure includes an A-type structure, the double strand constituting that part is usually an RNA double strand or an RNA and DNA double strand. For example, when the test nucleic acid is RNA, at least a part of the base sequence region, particularly the periphery of the detection domain, can be composed of DNA and / or RNA, and when the test nucleic acid is DNA, at least a part of the base sequence region, particularly the periphery of the detection domain, can be composed of RNA.

[0115] In this case, the length of the sequence surrounding the detection domain is not particularly limited, but for example, the sequence on the 3'-end and / or 5'-end of the detection domain can be 1 base or more, 2 bases or more, 3 bases or more, 4 bases or more, 5 bases or more, 6 bases or more, 7 bases or more, 8 bases or more, 9 bases or more, or 10 bases or more.

[0116] 1-3-6. Other embodiments Depending on the purpose, a nucleic acid molecule containing a shared base can be used as a nucleic acid base detection agent so that the base in the test nucleic acid functions as a detection domain. In this case, the nucleic acid base detection agent includes a nucleic acid molecule containing a base sequence region capable of hybridizing to the test nucleic acid, the base sequence region includes one shared base and includes a detection domain consisting of one actuator base and one indicator base adjacent thereto, the shared base forms a right-handed double-stranded structure together with the domain consisting of one actuator base contained in the test nucleic acid and one indicator base adjacent thereto, and the nucleic acid molecule has a configuration in which, when hybridized with the test nucleic acid, if the actuator base is other than a predetermined base, a base pair is formed between the shared base and the indicator base, and if the actuator base is a predetermined base, a base pair is formed between the shared base and the actuator base.

[0117] In this case, the presence of a given base can be detected by using a molecule that can bind to the structure in which the indicator base protrudes.

[0118] 1-3-7.Effects The nucleic acid base detector of the present invention makes it possible to simply and sensitively detect the type of base present at a specific position in a test nucleic acid.

[0119] The nucleic acid base detection agent of the present invention can be easily designed and synthesized if the type of the specific base to be detected and the base sequence around the specific base are known.In addition, since it can be designed to emit a detectable signal when the common base is a specific base, the risk of false positive is reduced by using the nucleic acid base detection agent of the present invention.In addition, the nucleic acid base detection agent of the present invention can detect the presence or absence of modification in base, which has been difficult to detect until now, with high sensitivity.

[0120] 2. Detection Composition 2-1. Overview The second aspect of the present invention is a detection composition. The detection composition of the present invention contains the nucleic acid base detection agent according to the first aspect as an active ingredient as an essential component. In addition, the detection composition may contain a carrier and / or a medium within a range that does not inhibit or suppress the detection ability of the nucleic acid base detection agent for a specific base. If necessary, the detection composition may further contain other active ingredients. By using the detection composition of the present invention, the presence of a specific base can be specifically detected. Each component will be specifically described below.

[0121] 2-2.Configuration 2-2-1. Components The detection composition of the present invention contains the nucleobase detection agent according to the first aspect as an essential active ingredient.

[0122] (1) Active ingredient The specific configuration of the nucleic acid base detection agent is not described here because it is described in detail in the first embodiment. The nucleic acid base detection agent used in the detection composition of this embodiment preferably contains an indicator base containing a detectable label.

[0123] The amount of active ingredient contained per unit amount in the composition depends on various conditions such as dosage form, type of label, use conditions, and use method.It is preferable that the active ingredient, the nucleic acid base detection agent, contains a sufficient amount of active ingredient to detect a specific base.Therefore, within the scope of the technical common knowledge in the field, each condition should be taken into consideration and determined so that the nucleic acid base detection agent contained in the detection composition of the present invention is an effective amount when used.

[0124] The amount of the active ingredient when the composition of the present invention is applied to a biological subject is not particularly limited. It is sufficient that an effective amount of the active ingredient is contained in a single application amount of the composition. However, when the amount of the composition that needs to be administered to the subject to obtain the effect of the active ingredient is large, it can be administered in several divided doses to reduce the burden on the subject. In this case, the amount of the active ingredient is sufficient as long as it contains an effective amount in total. The "effective amount" refers to an amount that is necessary to exert the function of the active ingredient and gives little or no harmful side effects to the subject to which it is applied. This effective amount may vary depending on various conditions such as the information of the subject, the application route, and the number of applications. Therefore, when the composition of this embodiment is used as a diagnostic pharmaceutical composition, the content of the active ingredient is ultimately determined by the judgment of a doctor, pharmacist, etc.

[0125] The type of organism in the present specification is not particularly limited, and includes all organisms including humans, and examples of organisms other than humans include various livestock, crops, poultry, pets, laboratory animals, and the like.

[0126] In this specification, "subject information" refers to various information related to the characteristics and condition of a subject. For example, when the subject is a human individual, information includes age, weight, sex, general health condition, presence or absence of disease, progression or severity of disease, drug sensitivity, presence or absence of concomitant drugs, and resistance to treatment.

[0127] The composition can include multiple nucleobase detectors, for example nucleobase detectors with different bases as predetermined bases, each of which can include a separately detectable indicator base.

[0128] (2) Carriers and media The carrier and medium used in the composition of the present invention are not particularly limited as long as they facilitate the use of the composition and the detection of a specific base, can maintain the detectability of the nucleic acid base detection agent as an active ingredient, and / or can control the action speed. For example, the hybridization solution described in the third embodiment may be used as a carrier or solvent.

[0129] (2-1) Carrier Specific examples of carriers that can be used in the present invention include protective agents, antioxidants, ultraviolet absorbers, emulsifiers, chelating agents, and pH buffers. The carriers may be mixed in advance or immediately before use.

[0130] The protective agent is expected to have an effect of reducing damage caused by ultraviolet rays, etc. Examples of the protective agent include skim milk, casein, gelatin, etc.

[0131] Examples of the antioxidant include phenol-based antioxidants, sulfur-based antioxidants, and phosphorus-based antioxidants.

[0132] Examples of ultraviolet absorbents include benzotriazole-based ultraviolet absorbents, benzophenone-based ultraviolet absorbents, other organic ultraviolet absorbents (e.g., para-aminobenzoic acid-based, oxybenzone-based, cinnamic acid-based, urocanic acid-based, anti-fading agents, etc.) and inorganic ultraviolet absorbents (e.g., titanium oxide, zinc oxide, silica, talc, and kaolin, etc.).

[0133] Examples of the emulsifier include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0134] Chelating agents include, for example, EDTA, EGTA, citric acid, salicylates, and the like. Examples of pH adjusters include alkaline agents such as sodium hydroxide and potassium hydroxide, and acid agents such as citric acid, sodium citrate, glycolic acid, and ascorbic acid.

[0135] When the composition of the present invention is applied to a biological subject, it is preferable to use a pharma- ceutically acceptable carrier as a carrier. The term "pharma-ceutically acceptable carrier" refers to additives that are commonly used in the field of formulation technology. For example, in addition to the above, excipients, binders, disintegrants, fillers, flow additive regulators, lubricants, human serum albumin, etc. can be mentioned.

[0136] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metallic salts, citric acid, tartaric acid, glycine, polyethylene glycol, Pluronic®, kaolin, silicic acid, or combinations thereof.

[0137] Binders include, for example, starch paste using vegetable starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, or combinations thereof.

[0138] Disintegrants include, for example, the above-mentioned starches, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearate monoglyceride, or salts thereof. Examples of fillers include petrolatum, the aforementioned sugars and / or calcium phosphate.

[0139] Examples of flow regulators and lubricants include silicates, talc, stearates or polyethylene glycol.

[0140] In addition to the above, if necessary, the composition may appropriately contain solubilizers, suspending agents, diluents, dispersing agents, surfactants, soothing agents, stabilizers, absorption promoters, bulking agents, moisturizing agents, humectants, adsorbents, flavoring agents, disintegration inhibitors, coating agents, colorants, preservatives, antiseptics, buffers, isotonicity agents, and the like that are commonly used in pharmaceutical compositions.

[0141] In addition, the detection composition of this embodiment may contain, for example, a delivery means, if necessary. The delivery means refers to a means that contains an active ingredient or other carriers, etc., delivers the contents, especially the active ingredient, to the target without decomposing it, and can control the distribution of the active ingredient in the sample (e.g., in the living body) in terms of time and quantity. Since the active ingredient of the detection composition of this embodiment is a nucleic acid, a delivery means can be used for the purpose of protecting it from decomposition by nuclease, transporting the membrane structure to the inside, etc. The type of delivery means used is not particularly limited. Examples include liposomes, polymeric micelles, and virus particles.

[0142] (2-2) Solvent The solvent used in the present invention is not particularly limited as long as it is capable of maintaining the detectability of the nucleic acid base detection agent, which is an active ingredient, and capable of maintaining the detectability of a predetermined base.

[0143] Solvents that can be used in the present invention include, for example, water (including aqueous solutions), buffers, or other aqueous solvents. Aqueous solutions include, for example, isotonic solutions containing physiological saline, glucose, and other auxiliary agents. Examples of auxiliary agents include, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride, as well as low-concentration nonionic surfactants (e.g., polysorbate 80, TM , HCO-60), polyoxyethylene sorbitan fatty acid esters, etc.

[0144] The pharmaceutical composition of the present invention may contain a pharma- ceutically acceptable solvent, if necessary. The term "pharma-ceutically acceptable solvent" refers to a solvent commonly used in the technical field of formulation.

[0145] (3) Other active ingredients In addition to the nucleic acid base detection agent described in the first aspect, the composition of the present invention may contain one or more other active ingredients having the same and / or different effects, provided that they do not affect the detectability of the nucleic acid molecule that constitutes the nucleic acid base detection agent.

[0146] The type of other active ingredient is not limited, and examples thereof include biomolecules or compounds such as nucleic acid molecules and peptides that have detectability for the same and / or different predetermined bases.

[0147] 2-2-2. Dosage form The dosage form of the detection composition of the present invention is not particularly limited as long as it is in a form that allows the active ingredient to be delivered to the target site without being inactivated in the sample.

[0148] In particular, when the sample is a biological subject, the specific dosage form will vary depending on the application method described below. Application methods can be broadly divided into parenteral administration and oral administration, so a dosage form appropriate for each administration method should be used.

[0149] For example, if the administration method is parenteral administration, the preferred dosage form is a liquid that can be administered directly to the target site or systemically via the circulatory system. A preferred example of a liquid is an injection. The injection can be formulated by appropriately combining the solvent, the excipient, emulsifier, suspending agent, surfactant, stabilizer, pH regulator, etc., and mixing them in a unit dose form required for generally accepted pharmaceutical practice.

[0150] The specific shape and size of each of the above dosage forms are not particularly limited as long as they are within the range of dosage forms known in the art. The method for producing the detection composition of the present invention may be prepared according to a conventional method in the art.

[0151] 2-3. Application method The application method of the composition of the present invention is not particularly limited. For example, it may be oral administration or parenteral administration. Oral administration is generally systemic administration, while parenteral administration can be further divided into systemic administration and local administration. Local administration includes, for example, intramuscular administration, subcutaneous administration, tissue administration, and organ administration, and systemic administration of parenteral administration includes intracirculatory administration, for example, intravenous administration (intravenous injection), intraarterial administration, and intralymphatic administration.

[0152] When the detection composition of the present invention is administered locally, it may be directly administered to the target site by injection, application, or the like. When the composition is administered systemically, it may be administered into the circulatory system by intravenous injection, or the like. The dosage may be any amount that is effective for the active ingredient to be effective. The effective amount is appropriately selected according to the information of the target, as described above.

[0153] 2-4. Detection target Various changes that affect the strength of base pairing can be detected. Specific examples of changes that affect the strength of base pairing include differences in modifications and mutations such as point mutations.

[0154] Examples of differences in modification include the presence or absence of modification, differences in the type of modification, etc. Examples of modifications include base modifications, sugar modifications, internucleoside bond modifications, and combinations thereof, etc. Examples of base modifications include methylation, deamination, thiolation, etc.

[0155] Examples of the mutation include point mutations. The type of point mutation is not particularly limited. Note that point mutations also include mutations that result in differences in modification. Examples of the point mutation include single nucleotide polymorphisms.

[0156] The detection composition of the present invention can be, for example, a composition for detecting a difference in modification, a composition for detecting the presence or absence of modification, a composition for detecting a point mutation, a composition for detecting a single nucleotide polymorphism, etc., depending on the type of the predetermined base to be detected. According to the composition of the present invention, a nucleic acid or a cell containing a predetermined base to be detected can be detected. Therefore, the composition of the present invention can be, for example, a composition for detecting a nucleic acid of interest, a composition for detecting a cell of interest, etc.

[0157] 2-5.Effects The detection composition of the present invention can detect the presence of a specific base in a sample. For example, a mutation (e.g., single nucleotide polymorphism) in an individual from which a biological sample is derived can be detected. In addition, for example, by designing a nucleic acid molecule so as to be able to detect a mutation specific to a cell (e.g., a cancer cell) to be treated, the detection composition of the present invention can be used to visualize the cell during treatment.

[0158] 3. Kit 3-1. Overview The third aspect of the present invention is a kit. The kit of the present invention contains the nucleic acid base detection agent according to the first aspect and / or the detection composition according to the second aspect as an active ingredient, and is configured to detect a predetermined base present in a sample. By using the kit of the present invention, the presence of the predetermined base can be specifically detected.

[0159] 3-2.Configuration The kit of the present invention is characterized by including a nucleic acid base detecting agent and / or a composition for detection as an essential component.

[0160] The specific configuration of the nucleic acid base detection agent is not described here because it is described in detail in the first embodiment, and the specific configuration of the detection composition is not described here because it is described in detail in the second embodiment.

[0161] The kit of the present invention may include a container. The material of the container is not limited as long as it is made of a material that does not contaminate the contents or is not contaminated by the contents. For example, the container may be made of a material such as plastics such as polypropylene or polystyrene, glass, or paper with a specially coated surface.

[0162] In the kit of the present invention, the nucleic acid base detector and / or the detection composition can be contained in a container. The kit of the present invention may include multiple types of nucleic acid base detectors capable of detecting different predetermined bases, in which case the multiple types of nucleic acid base detectors can be contained in one or more containers.

[0163] The kit of the present invention may further contain a hybridization solution. In the kit of the present invention, the hybridization solution can be contained in the same or a different container from the nucleic acid base detection agent or the like.

[0164] In the present specification, the hybridization solution is not particularly limited as long as it is a solution in which the nucleic acid molecules contained in the nucleic acid base detection agent or the like can form a double-stranded structure. The hybridization solution may be, for example, a solution containing a salt and / or a buffer. Here, examples of the salt include lithium chloride, sodium nitrate, potassium acetate, ammonium formate, magnesium sulfate, calcium sulfate, strontium sulfate, and barium sulfate.

[0165] Examples of the buffer include sodium cacodylate, potassium cacodylate, sodium phosphate, disodium hydrogen phosphate, BIS-TRIS, HEPES, TRIS, MES, MOPS, sodium citrate, ammonium iron citrate, succinic acid, tricine, and sodium acetate.

[0166] The concentration of the salt in the hybridization solution is not particularly limited. For example, it can be 1 mM to 10 M, 1 mM to 1 M, 1 mM to 500 mM, or 1 mM to 200 mM. The concentration of the buffer in the hybridization solution is not particularly limited. For example, it can be 1 mM to 1 M, 10 mM to 100 mM, 10 mM to 50 mM, or 10 mM to 30 mM. The pH of the hybridization solution is not particularly limited, but can be, for example, pH ≤ 11, specifically 5 < pH < 8, or 8 ≤ pH ≤ 11. The hybridization solution may be, for example, a solution containing sodium chloride and sodium cacodylate, such as a solution containing 10 mM sodium cacodylate (pH 7) and 100 mM sodium chloride.

[0167] The kit of the present invention can include means for contacting a test sample with a nucleic acid molecule, such as those used in the contacting step of the fifth aspect.

[0168] The kit of the present invention may comprise a means capable of detecting the indicated base of a nucleic acid molecule, for example, as used in the detection step of the fifth aspect. Detectable means refers to, for example, a reagent that can be used for detection and / or a detection device that can be used for detecting the resulting signal.

[0169] Examples of reagents that can be used for detection include reagents capable of detecting a form in which the indicator base protrudes outward from the double-stranded structure, etc. Examples of reagents capable of detecting a form in which the indicator base protrudes outward from the double-stranded structure include reagents containing a molecule capable of binding to the indicator base, and reagents containing a molecule capable of binding to all or a part of the three-dimensional structure in which the indicator base protrudes outward from the double-stranded structure, etc.

[0170] The molecule that can be bound is not particularly limited, but examples include antibodies, antigen-binding fragments thereof, and aptamers such as nucleic acid aptamers and peptide aptamers.

[0171] The detection device is not particularly limited. The detection method can be appropriately selected according to the label and reagent used, and the properties of the test sample, and the like, and the device required for the detection method can be used. For example, when a fluorescent dye or luminescent substance is used, it can be detected, for example, by visual inspection, using a microscope (e.g., a fluorescent microscope, etc.), using a detector (e.g., fluorescence activated cell sorting (FACS), a luminescence photometer, an absorption photometer, etc.), or by a combination thereof. When a non-colored small molecule that acts as an enzyme substrate or antigen is used as a label, it can be detected, for example, after treatment such as enzyme treatment, by the same detection method as when a fluorescent dye or luminescent substance is used. When a radioisotope is used as a label, it can be detected, for example, by autoradiography, a scintillation counter, positron emission tomography (PET), or a combination thereof.

[0172] The kit of the present invention may include a control sample containing a known amount of a predetermined base, for example, as used in the determination step of the fifth embodiment. The kit may include one or more control samples. In addition, when the kit includes a nucleic acid molecule capable of detecting multiple types of predetermined bases, the kit may include a control sample for each of the predetermined bases.

[0173] The kit of the present invention may include an indicator for determining the presence of a predetermined base in a test sample, such as that used in the determination step of the fifth embodiment. The kit may include one or more indicators. In addition, when the kit includes a nucleic acid molecule capable of detecting multiple types of predetermined bases, the kit may include a control sample for each of the predetermined bases. In addition, the kit may include both a control sample and an indicator.

[0174] The kit of the present invention may include an instruction manual as needed. The kit of the present invention may include an application means such as a syringe, an injection needle, a sprayer, etc. as needed.

[0175] The kit of the present invention can be used as a detection kit for a specific base to detect a specific base in a test sample. In addition, depending on the type of the specific base to be detected, it can be, for example, a detection kit for modification difference, a detection kit for the presence or absence of modification, a detection kit for point mutation, a detection kit for single nucleotide polymorphism, etc. According to the kit of the present invention, it is possible to detect a nucleic acid or a cell containing a specific base to be detected. Therefore, the kit of the present invention can be, for example, a detection kit for a nucleic acid of interest, a detection kit for a cell of interest, etc.

[0176] 4. Devices 4-1. Overview The fourth aspect of the present invention is a device. The device of the present invention contains the nucleic acid base detection agent described in the first aspect and / or the detection composition described in the second aspect as an essential component in the reagent part, and can detect the presence of a predetermined base. By using the detection device of the present invention, the presence of a predetermined base can be specifically detected.

[0177] 4-2.Configuration 4-2-1. Components The device of the present invention includes a sample receiving section, a reagent section, a reaction section, and a presentation section as essential components, and a development section as an optional component. The development section may further include a labeling means. Each component will be described below.

[0178] 4-2-2. Sample receiving section The "sample receiving section" is configured to receive a sample that may contain a predetermined base. The sample received in this section is not particularly limited, but includes test samples and control samples used in the detection method described in the fifth aspect. The amount of sample received is not limited as long as it is equal to or greater than the minimum amount required to detect the predetermined base. It can be appropriately determined depending on the sensitivity of the nucleic acid molecule contained in the reagent section, the composition and form of the test sample, etc.

[0179] The sample receiving portion may be in the form of a well-like container capable of storing the received sample. In this case, the material of the sample receiving portion is not limited as long as it is made of a material that does not contaminate the sample or is not contaminated by the sample. Examples of materials include plastics such as polypropylene and polystyrene, glass, and paper with a specially coated surface. The sample receiving portion may also be in the form of a sheet or rod that easily absorbs the sample. In this case, it may be made of a material that easily absorbs the sample, such as a nonwoven fabric or a filter.

[0180] The sample and the method of addition are not particularly limited. For example, if the sample is a liquid, it may be dropped onto or impregnated into the sample receiving part. If the sample is a solid, the sample suspended in a liquid may be dropped onto or impregnated into the sample receiving part, or a moistened sample receiving part may be brought into contact with the sample. If the sample is a gas or aerosol, the sample absorbed in a liquid may be dropped onto or impregnated into the sample receiving part, or a moistened sample receiving part may be brought into contact with the sample.

[0181] 4-2-3.Reagent section The "reagent part" is configured to contain the nucleic acid base detection agent according to the first aspect and / or the detection composition according to the second aspect. The nucleic acid molecule contained in the nucleic acid base detection agent and / or the detection composition may be immobilized on the surface of a carrier in the reagent part, or may be in a free state within the reagent part.

[0182] The amount of the nucleic acid molecule contained in the reagent portion is not limited as long as it is equal to or greater than the minimum amount necessary to detect a predetermined base contained in a sample.

[0183] The reagent section may be located in a different position from the sample receiving section in the device. In this case, the sample received in the sample receiving section and the nucleic acid molecules contained in the reagent section are configured to pass through respective flow paths and be delivered to the reaction section described below. The sample receiving section and the reagent section may also be located in the same position to serve as a sample receiving section / reagent section (sample receiving section / reagent section). In this case, the sample receiving section contains nucleic acid molecules, and the received sample immediately reacts with the nucleic acid molecules. Therefore, in this case, the sample receiving section / reagent section can also serve as the reaction section described next.

[0184] 4-2-4.Reaction section The "reaction section" is configured to function as a site where a predetermined base contained in a sample can react with a nucleic acid molecule. There are no particular limitations on the configuration of the reaction section, so long as it is a section that can provide conditions for the reaction between the two to proceed.

[0185] 4-2-5. Development section The "development section" is a section that can be a flow path through which reactants generated in the reaction section move to the presentation section described below, and is an optional component in the device of the present invention. When the reaction section and the presentation section are installed in different locations in the device of the present invention, the development section can function as a section that connects the two sections. The structure of the development section can be, but is not limited to, a groove-like or tubular flow path.

[0186] (1) Marking means The development section may include a labeling means. The "labeling means" is a means for labeling the reactant produced in the reaction section before the reactant reaches the presentation section. The labeling means is configured so that the reactant is labeled with a labeling substance. The specific configuration of the labeling means is not particularly limited. For example, the reagents usable for detection described in the section on the kit of the third embodiment can be used. The labeling means may be contained in the reaction section or in the presentation section described below.

[0187] (2) Removal means The development section may include a removal means. The "removal means" is a means for removing nucleic acid molecules not bound to test nucleic acids and / or nucleic acids not bound to nucleic acid molecules, etc., before the reaction product generated in the reaction section reaches the presentation section. The removal means is configured to remove unreacted nucleic acid molecules, other nucleic acids, and / or contaminants, etc., in the reaction product. The specific configuration of the removal means is not particularly limited. For example, a membrane structure or adsorbent capable of removing molecules capable of binding to nucleic acid molecules not bound to test nucleic acids, nucleic acids including test nucleic acids not reacted with nucleic acid molecules, and other minute contaminants, can be used. The removal means may be included in the reaction section or the presentation section described below.

[0188] 4-2-6. Presentation section The "presentation unit" is configured to present the detection result in the reaction unit, i.e., the amount of reactant produced in the reaction unit and the presence or absence of reactant. Specifically, for example, the presentment unit may be configured to present the success or failure of detection in a visually recognizable manner, or the content of a predetermined base or the type of detected base in a visually recognizable manner.

[0189] The means of presentation is not particularly limited. For example, in the case of using a color reaction, the color development may be presented at any location on the device, and in the case of using a fluorescence or luminescence reaction, the fluorescence or luminescence may be presented at any location on the device. Alternatively, the level or type of the reaction may be presented as a numerical value, a character, or the like.

[0190] Specific examples of devices of this embodiment include test strips and the like used in immunochromatography known in the art, test papers and the like in which nucleic acid molecules are impregnated on filter paper or the like, ion concentration meters and the like using sensitive membranes or the like, and detector tubes, digital gas meters and the like.

[0191] The device of the present invention can be used as a predetermined base detection device for detecting a predetermined base in a test sample or a target nucleic acid detection device. In addition, depending on the type of the target base, it can be, for example, a device for detecting the difference in modification, a device for detecting the presence or absence of modification, a device for detecting point mutations, a device for detecting single nucleotide polymorphisms, etc. According to the device of the present invention, it is possible to detect nucleic acids or cells containing the target base. Therefore, the method of the present invention can be, for example, a device for detecting a target nucleic acid, a device for detecting a target cell, etc.

[0192] 5. Detection Method 5-1. Overview The fifth aspect of the present invention is a method for detecting a target nucleic acid containing a specific base as a covalent base. According to this method, the presence or absence of a target nucleic acid containing a specific base as a covalent base can be detected using the nucleic acid base detection agent described in the first aspect and / or the detection composition described in the second aspect.

[0193] 5-2. Method The method of this embodiment includes a contact step, a detection step, and a determination step as essential steps, and includes a suspension step and a nucleic acid concentration step as optional steps. Each step will be specifically described below.

[0194] 5-2-1. Suspension process This step is an optional step in which the test sample is suspended in a liquid. When the test sample is not a liquid, this step can be performed, whereas when the test sample is in a form selected from the group consisting of a liquid, a semisolid, and an aerosol, for example, this step does not need to be performed.

[0195] As used herein, the term "test sample" refers to a sample that may contain a target nucleic acid that contains a specific base as a covalent base. Test samples that can be used herein include, for example, biological samples such as body fluids and cells, and environmental samples such as air, soil, and water.

[0196] 5-2-2. Nucleic acid concentration step This step is an optional step for concentrating nucleic acids in a test sample and can be performed simultaneously with or after the contacting step.

[0197] The concentration method used in this step is not particularly limited. For example, a solute may be concentrated non-selectively regardless of whether it is a nucleic acid or not, a nucleic acid may be concentrated non-selectively regardless of the sequence of the nucleic acid, or a nucleic acid having a specific sequence may be selectively concentrated.

[0198] As a method for non-selective concentration, any method used for concentrating nucleic acid molecules can be used, such as ultrafiltration, ion exchange chromatography, diatomaceous earth method, size exclusion chromatography, and methods using spin columns or magnetic beads.

[0199] Methods for selectively enriching nucleic acids having a specific sequence include amplification by PCR and affinity chromatography using nucleic acids that can hybridize to a specific sequence.

[0200] This process can be carried out multiple times, either using the same method each time or using a different method each time.

[0201] 5-2-3. Contact process This step is a step of contacting a test sample with the nucleic acid base detection agent according to the first aspect and / or the composition for detection according to the second aspect. When a suspension step is performed, this step can be performed simultaneously with or after the suspension step.

[0202] The contact method used in this step is not particularly limited. In the present invention, at least one of the test sample and the nucleic acid base detection agent and / or the detection composition is liquid. Therefore, as long as the method can directly contact a solid with a liquid, or a liquid with a liquid, there is no particular limitation. Specifically, for example, the contact can be achieved by immersing the solid in the liquid, spraying, spraying or applying the liquid to the solid, dropping or mixing the liquid into the liquid, or a combination thereof.

[0203] It is sufficient that a part of the test sample is contacted with the nucleic acid base detection agent and / or the detection composition, and it is not necessary that the whole of the test sample is contacted.Specifically, for example, when the test sample is a large solid, the nucleic acid base detection agent and / or the detection composition can be contacted with a part of the test sample to detect the target nucleic acid contained in the test sample.In addition, for example, when the test sample is a cell sample or tissue sample, the nucleic acid base detection agent and / or the detection composition can be contacted by spraying, applying, or injecting the test sample.

[0204] The contact time is not particularly limited. For example, it can be determined in relation to the type of test sample, contact conditions, etc. Specifically, it can be, for example, 1 second or more, 3 seconds or more, 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, or 30 minutes or more.

[0205] The contact can be carried out multiple times. In this case, the composition of the nucleic acid base detection agent and / or the detection composition, the contact time, and other conditions may be changed for each contact, or the same conditions used in the previous contact may be used as they are.

[0206] 5-2-4.Detection process This step is a step of detecting the protrusion of the indicator base from the double-stranded structure. This step can be performed simultaneously with or after the contacting step.

[0207] The detection method used in this step is not particularly limited as long as it is a method capable of detecting the state in which the indicator base protrudes outward from the double-stranded structure. For example, the SPR method, turbidimetric method, colorimetric method, fluorescent method, or a combination thereof may be used.

[0208] SPR (surface plasmon resonance) refers to a phenomenon in which the reflected light intensity is significantly attenuated at a specific angle of incidence (resonance angle) when a metal thin film is irradiated with laser light. The SPR method is a measurement method that utilizes this phenomenon, and can measure adsorbates on the surface of the metal thin film, which is the sensor part, with high sensitivity. In the present invention, for example, the nucleic acid molecule of the present invention is immobilized on the surface of the metal thin film in advance, a sample is passed over the surface of the metal thin film, and the target nucleic acid in the sample can be detected by detecting the difference in the amount of adsorbates on the metal surface before and after the sample has passed, which is caused by the binding between the nucleic acid molecule and the target nucleic acid. The SPR method includes a displacement method, an indirect competitive method, and the like, and any of these may be used.

[0209] The turbidimetric method is a method for measuring the amount of a substance in a solution by irradiating a solution with light and optically measuring the attenuation of the scattered light caused by a substance suspended in the solution or the transmitted light passing through the solution using an absorptiometer or the like or by visual inspection. In the present invention, the target nucleic acid in a sample can be quantitatively detected by measuring the absorbance before and after adding the nucleic acid molecule of the present invention to the sample, or before and after adding the sample to the nucleic acid base detection agent and / or detection composition of the present invention.

[0210] The colorimetric method is a method for measuring the amount of a substance in a reaction product by optically measuring the intensity or wavelength of transmitted or reflected light in the colored reaction product. This method can be performed using a colorimeter or the like, or visually. In the present invention, the target nucleic acid in a sample can be quantitatively detected by measuring the absorbance and / or color before and after adding the nucleic acid molecule of the present invention to the sample, or before and after adding the sample to the nucleic acid base detection agent and / or detection composition of the present invention.

[0211] The fluorescence method is a method for measuring the amount of a substance in a reaction product by irradiating a reaction product containing a fluorescent substance with excitation light and optically measuring the intensity and wavelength of the emitted fluorescence. This method can be performed using a fluorescence detector or the like, or visually. In the present invention, the target nucleic acid in a sample can be quantitatively detected by measuring the intensity and / or wavelength of the fluorescence before and after adding the nucleic acid molecule of the present invention to the sample, or before and after adding the sample to the nucleic acid base detection agent and / or detection composition of the present invention.

[0212] In addition, detection can be performed by using a form in which the display base protrudes outward from the double-stranded structure in combination with a detectable molecule. For example, a method that applies the sandwich method of the ELISA method may be used. In this method, for example, first, the nucleic acid molecule of the present invention is fixed to a solid phase carrier, and then a sample is added to bind the target nucleic acid and the nucleic acid molecule present in the sample. Then, after washing the sample, the molecule described in the third embodiment is added to bind to the complex of the target nucleic acid and the nucleic acid molecule. After washing, the target nucleic acid in the sample can be detected by detecting the molecule bound to the complex using a secondary antibody that is appropriately labeled. As the solid phase carrier, an insoluble carrier in the form of beads, microplates, test tubes, sticks, test strips, etc. made of materials such as polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, latex, gelatin, agarose, cellulose, sepharose, glass, metal, ceramics, or magnetic material can be used.

[0213] 5-2-5. Judgment process This step is a step of determining that the target nucleic acid is present in the test sample when the overhang of the indicated base is detected. This step can be performed simultaneously with or after the detection step.

[0214] Whether or not an overhang of the indicated base is detected can be determined using any method. For example, the determination may be made relatively using a control sample or the like, or may be made absolutely based on the results obtained in the detection step.

[0215] In the case of relatively determining, specifically, for example, when the result of the detection step in the test sample shows that the overhang of the indicated base occurs more frequently than the control sample, the test sample can be determined to contain the target nucleic acid. As the control sample, a sample that clearly does not contain the target nucleic acid, or a sample that clearly contains a similar amount of nucleic acid and contains a known amount of the target nucleic acid, etc., can be used. Whether or not the result shows that the overhang of the indicated base occurs more frequently can be determined, for example, qualitatively, or quantitatively, for example, by using a statistical method, based on the presence or absence of a significant difference.

[0216] In this specification, "significant" means statistically significant. Statistically significant means that there is a significant difference between the measured value of the subject and the control value when the difference between them is statistically processed. For example, the risk rate (significance level) of the obtained value is small, specifically, less than 5% (p<0.05), less than 1% (p<0.01), and less than 0.1% (p<0.001). The "p (value)" shown here indicates the probability that the test statistic will coincidentally become that value in a distribution based on the null hypothesis in a statistical test. Therefore, the smaller "p" is, the lower the probability that the test statistic will become that value, and the easier it is to reject the null hypothesis. The test method for statistical processing is not particularly limited, and any known test method capable of determining the presence or absence of significance may be used appropriately. For example, Student's t-test, paired Student's t-test, Welch's t-test, Wilcoxon rank sum test, analysis of variance, Tukey post-hoc test, etc. can be used, but are not limited thereto.

[0217] Alternatively, the determination can be made using one or more samples containing a known amount of the target nucleic acid as a control sample, in which case the amount of the target nucleic acid contained in the test sample can be estimated by this step.

[0218] When making an absolute determination, it is possible to determine whether or not the target nucleic acid is contained in the test sample, or the amount thereof, from the results obtained in the detection step, for example, based on a specific indicator.

[0219] For example, when the signal changes quantitatively due to the protrusion of the indicator base, the relationship between the signal intensity and the amount of the target nucleic acid can be used as an index to make a judgment from the signal intensity observed in the test sample. Specifically, for example, when the index is a threshold for distinguishing the presence or absence of the target nucleic acid, when a signal with an intensity exceeding the threshold is obtained from the test sample, it can be judged that the test sample contains the target nucleic acid or contains a certain amount of the target nucleic acid.

[0220] When a quantitative value is used for the determination, a value standardized using other indices can be used as the value used for the determination. Specifically, for example, a value standardized with respect to the amount of nucleic acid in the test sample or a value standardized with respect to a control sample can be used. In this case, the measurement of the amount of nucleic acid used is not particularly limited, but for example, a method based on absorbance or electrophoresis can be used.

[0221] Furthermore, for example, when the signal is qualitatively changed by the protrusion of the indicator base (for example, when the color is changed, etc.), the relationship between the quality of the signal and the amount of the target nucleic acid can be used as an index to make a judgment from the signal observed in the test sample. Specifically, for example, when the indicator is a color sample for distinguishing the presence or absence of the target nucleic acid, when the signal obtained from the test sample changes beyond that color, it can be judged that the test sample contains the target nucleic acid.

[0222] A plurality of criteria can be used for the determination. Specifically, for example, a criterion for determining the type of unmodified base of a given base and a criterion for determining the presence or absence of modification of a given base can be used. Also, for example, a criterion for each type of base can be used.

[0223] The method of the present invention can be, for example, a method for detecting a difference in modification, a method for detecting the presence or absence of modification, a method for detecting a point mutation, a method for detecting a single nucleotide polymorphism, etc., depending on the type of the predetermined base to be detected. According to the method of the present invention, a nucleic acid or a cell containing the predetermined base to be detected can be detected. Therefore, the method of the present invention can be, for example, a method for detecting a nucleic acid of interest, a method for detecting a cell of interest, etc.

[0224] When the method of the present invention is a method for detecting a target cell, if an overhang of the indicator base is detected in this step, it is determined that a target cell is present in the test sample. EXAMPLES

[0225] <Example 1: Design of nucleic acid base detector and verification of its function (1)> (the purpose) A nucleic acid base detection agent composed of nucleic acids is designed and its ability to detect the types of bases is verified.

[0226] (method) 1. Design and synthesis of nucleic acid molecules As the test nucleic acid, a common base X 1 An RNA molecule was designed having the following structure: (5'-ACUCUCCUCGX 1 CUCUUCAUCAUCAUGU-3': SEQ ID NO: 1; FIG. 2A, left strand), where: 1 We designed an RNA molecule in which the amino acid sequence is adenine (RNA-A: sequence number 2), guanine (RNA-G: sequence number 3), cytosine (RNA-C: sequence number 4), and uracil (RNA-U: sequence number 5).

[0227] As a detection nucleic acid molecule, a working base Y 1 A molecule was designed with the display base 2AP (2-Aminopurine) (5'-CATGATGATGAAGAGA * Y 1 CGAGGAGAGT-3'(A * 2-aminopurine): SEQ ID NO: 6; FIG. 2A, right strand), where Y 1 Depending on the type of Y1 We designed a DNA molecule in which the amino acid is thymine (adenine detection nucleic acid molecule: sequence number 7), a DNA molecule in which the amino acid is guanine (cytosine detection nucleic acid molecule: sequence number 8), an RNA molecule in which the amino acid is cytosine (guanine detection nucleic acid molecule: sequence number 9), and an RNA molecule in which the amino acid is adenine (uracil detection nucleic acid molecule: sequence number 10).

[0228] The test nucleic acid and the detector nucleic acid molecule form a heterodimer, and the working base X 1 and a sensing domain consisting of the representative base 2AP and the shared base X 1 are designed to form a base triplet.

[0229] With this design, when the shared base is a base other than the specified base, the 3D structure shown in Figure 1A is formed, and X 1 On the other hand, when the shared base is a specific base, the structure shown in Figure 1B is formed, and the X:2AP base pair is formed. 1 :Y 1 It is believed that a base pair of the formula is formed. As a result, when the shared base is a specific base, it is expected that 2AP will protrude and its fluorescent signal will be strongly observed.

[0230] The nucleic acid molecules were synthesized using an automatic nucleic acid synthesizer NTS-M2-MX (Nihon Techno Service Co, Ltd). In this case, 2-aminopurine-CE phosphoramidite (Glen Research) was used as the phosphoramidite of the nucleotide containing 2-aminopurine. The synthesized sample was purified by gel filtration using Illustra NAP-10 columns (Cytiva). After purification, 20% denaturing polyacrylamide gel electrophoresis containing 7M urea was performed to confirm that the sample was correctly synthesized with high purity.

[0231] 2. Fluorescence Intensity Measurement A solution containing 100 mM sodium nitrate and 10 mM 3-morpholinopropanesulfonic acid (MOPS: pH 7) was used as a fluorescence measurement buffer, and the synthesized detection nucleic acid molecule was added to a final concentration of 0.01 mM to prepare a fluorescence measurement buffer as a test solution.

[0232] The measurement conditions for the fluorescence spectrum (excitation wavelength and range of fluorescence wavelengths to be detected) were determined by measuring the 3D fluorescence spectrum of a test solution containing each detection nucleic acid molecule using an FP-8300 (Jasco) and identifying the maximum excitation wavelength and maximum fluorescence wavelength.

[0233] The following measurement conditions were used in the subsequent experiments: Excitation wavelength: 305 nm Detection wavelength: 330nm~470nm.

[0234] Furthermore, the rate of change in fluorescence intensity ΔF when various test nucleic acids were added to the test solution to a final concentration of 0.01 mM was calculated using the following formula, and the detection ability of the detection nucleic acid molecule was evaluated. ΔF = (FF 0 ) / F (F: Fluorescence intensity at peak wavelength in the test solution after addition of the test nucleic acid, F 0 : Fluorescence intensity at peak wavelength in test solution without added test nucleic acid)

[0235] As the peak wavelength of each detection nucleic acid molecule, the following values ​​were used based on the results of measuring the fluorescence spectrum using each test solution without adding the test nucleic acid: Adenine detection nucleic acid molecule: 375 nm Nucleic acid molecules for detecting cytosine: 370 nm Guanine detection nucleic acid molecule: 370 nm Nucleic acid molecules for detecting uracil: 360 nm. These steps were carried out at room temperature.

[0236] (result) The results are shown in Figures 3 to 6. Regardless of which detection nucleic acid was used, the fluorescence intensity increased significantly when a test nucleic acid having a specific base to be detected was added to the test solution. The change in fluorescence intensity was significantly greater than when the shared base was a base other than the specific base.

[0237] This demonstrated that the detection nucleic acid was capable of specifically detecting the type of base.

[0238] <Example 2: Design of nucleic acid base detector and verification of its function (2)> (the purpose) We design a nucleic acid base detection agent composed of nucleic acids and verify its ability to detect the presence or absence of base modifications.

[0239] (method) The design and synthesis of test nucleic acids and nucleic acid molecules, and the measurement of fluorescence intensity were carried out in the same manner as in Example 1, except that the nucleic acid sequences were partially different.

[0240] The test nucleic acid is a common base X 2 An RNA molecule was designed having the following structure: (5'-ACUCUCCUCGX 2 CUCUUCAUCAUCAUGU-3': SEQ ID NO: 11; FIG. 2B, left strand) was designed. 2 In addition to the RNA molecule where X is an adenine (RNA-A: SEQ ID NO: 2), and the RNA molecule where X is a cytosine (RNA-C: SEQ ID NO: 4), 2 RNA molecules in which the amino acid is 6-methyladenine (RNA-m 6 A: SEQ ID NO: 12), an RNA molecule that is 5-methylcytosine (RNA-m 5 C: sequence number 13) were designed.

[0241] The detection nucleic acid molecule includes the working base Y 2 A molecule was designed with the display base 2AP (2-Aminopurine) (5'-CATGATGATGAAGAGA * Y 2 CGAGGAGAGT-3'(A * 2-aminopurine): SEQ ID NO: 14; FIG. 2B, right strand), where Y2 Depending on the type of Y 2 A DNA molecule in which the amino acid residue is thymine (adenine-detecting nucleic acid molecule: SEQ ID NO: 7) and a DNA molecule in which the amino acid residue is guanine (cytosine-detecting nucleic acid molecule: SEQ ID NO: 8) were designed.

[0242] (result) The results are shown in Figures 7 and 8. Regardless of which detection nucleic acid was used, the fluorescence intensity increased significantly when the test nucleic acid having the specified methyl-modified base was added to the test solution. The change in fluorescence intensity was significantly greater than when the shared base was an unmodified base.

[0243] This demonstrates that the detecting nucleic acid of the present invention is capable of detecting not only the type of base but also the presence or absence of modification.

Claims

1. A nucleobase detection agent for detecting a predetermined base in a test nucleic acid, wherein the nucleobase detection agent includes a nucleic acid molecule containing a base sequence region capable of hybridizing to the test nucleic acid, the base sequence region includes a detection domain consisting of one operative base and one display base adjacent thereto, when the nucleic acid molecule hybridizes with the test nucleic acid, the detection domain forms a right-handed double-stranded structure together with one shared base contained in the test nucleic acid, when the shared base is other than the predetermined base, a base pair of the shared base and the display base is formed, when the shared base is the predetermined base, a base pair of the shared base and the operative base is formed, the nucleobase detection agent.

2. The nucleobase detection agent according to claim 1, wherein the base sequence region has a length of 6 bases or more.

3. The nucleobase detection agent according to claim 1, wherein the nucleic acid molecule includes a plurality of the detection domains.

4. The nucleobase detection agent according to claim 1, wherein the predetermined base is one or more bases selected from the group consisting of adenine, thymine, guanine, cytosine, uracil, 6-methyladenine, 4-methylthymine, 6-methylguanine, 7-methylguanine, 5-methylcytosine, and 5-methyluracil.

5. A composition for detecting a predetermined base, comprising the nucleobase detection agent according to claim 1.

6. A composition for detecting a point mutation, comprising the nucleobase detection agent according to claim 1.

7. The detection composition according to claim 6, wherein the point mutation is a single nucleotide polymorphism.

8. The detection composition according to any one of claims 5 to 7, comprising a plurality of types of nucleobase detection agents.

9. A kit for detecting a predetermined base, comprising the nucleobase detection agent according to claim 1 and / or the detection composition according to claim 5.

10. A device for detecting a predetermined base, comprising the nucleobase detection agent according to claim 1 and / or the detection composition according to claim 5.

11. A method for detecting a target nucleic acid containing a predetermined base as a shared base, comprising: a contacting step of contacting a test sample containing a nucleic acid with the nucleobase detection agent according to claim 1 and / or the detection composition according to claim 5; a detecting step of detecting the protrusion of the display base from the double-stranded structure; and a determining step of determining that the target nucleic acid is present in the test sample when the protrusion of the display base is detected. The detection method.

12. The detection method according to claim 11, wherein the test sample is in a form selected from the group consisting of a liquid, a semi-solid, and an aerosol.

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