Mismatch recognition molecule, mismatch detection method, disease diagnosis method, and method for producing mismatch recognition molecule

Abstract

To provide a molecule capable of recognizing a cytosine-cytosine mismatch in DNA.SOLUTION: A solution of FcDANP represented by the chemical formula below, and three solutions each obtained by mixing FcDANP with one of three DNAs having different base sequences, are measured using an ultraviolet-visible spectrophotometer. In the wavelength range of approximately 375-400 nm, the sample containing FcDANP and the DNA having a C-C mismatch exhibits a significantly higher absorption intensity than the other samples.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a novel mismatch recognition molecule that specifically binds to a specific mismatch that occurs in a nucleic acid and has a recognition function such as fluorescence; a mismatch detection method that utilizes the recognition function of the mismatch recognition molecule to detect a specific mismatch that occurs in a nucleic acid; a disease diagnosis method that utilizes the mismatch recognition molecule and the mismatch detection method; and a method for producing the mismatch recognition molecule. [Background technology]

[0002] When nucleic acids such as DNA and RNA hybridize to form a double strand, the combination of bases that form pairs is fixed; specifically, guanine (G) pairs with cytosine (C), and adenine (A) pairs with thymine (T). Therefore, when nucleic acids are hybridized, all bases usually form pairs as described above, but in some cases, some of the base sequences in the nucleic acid cannot form such pairs. When single-stranded nucleic acids hybridize to form a double strand, base pairs that cannot form normal base pairs are generally referred to as mismatches.

[0003] In recent years, research into mismatches in genomic sequences has progressed. A typical example of a mismatch in a genomic sequence is a single nucleotide polymorphism (SNP), in which a single base in a nucleic acid sequence differs from the normal base due to a mutation in the nucleic acid. Recent research findings suggest that SNPs are one of the causes of genetic diseases and one of the factors that contribute to individual differences among organisms. Furthermore, in the pharmaceutical field, it is known that drugs developed to treat certain diseases are effective in some patients, while others are not. If SNPs could be used to distinguish between these patients with different drug responses, more rational and efficient drug dosing would be possible, potentially reducing side effects, particularly in the case of anticancer drugs.

[0004] Various techniques have been proposed for detecting mismatches. Patent documents 1 to 3 listed below disclose inventions that utilize compounds that form pseudo-base pairs with mismatches, and patent documents 4 and 5 disclose inventions that detect mismatches by hybridizing them with other single-stranded nucleic acids. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2004-261083 [Patent Document 2] Japanese Patent Application Laid-Open No. 2004-275179 [Patent Document 3] Japanese Patent Application Laid-Open No. 2006-094725 [Patent Document 4] WO2016 / 098595 publication [Patent Document 5] Japanese Patent Application Laid-Open No. 2007-312621 Summary of the Invention [Problem to be solved by the invention]

[0006] The inventions disclosed in Patent Documents 1 to 3 have the problem that a compound that forms a relatively strong bond with a nucleic acid containing a mismatch needs to be immobilized on a column, an SPR substrate, etc., which requires a lot of time and expensive equipment. Also, the inventions disclosed in Patent Documents 4 and 5 have the problem that it is time-consuming to hybridize the target nucleic acid with another single-stranded nucleic acid, and that it is necessary to redesign the detection nucleic acid depending on the base sequence near the target mismatch.

[0007] Furthermore, even if a molecule that specifically binds to the type of mismatch to be detected is obtained, in order to reliably detect the molecule specifically bound to the mismatch, it would be preferable for the molecule to have some characteristic (special function) useful for detection, such as the ability to emit fluorescence. However, molecules that specifically bind to DNA mismatches and have characteristics useful for detection, such as fluorescence, have not been known until now, including the inventions disclosed in Patent Documents 1 to 5. This is thought to be because, depending on the base sequence of the DNA, covalently binding a molecule synthesized with the aim of imparting characteristics such as fluorescence to a mismatch on DNA often takes a long time, even using specialized equipment. In addition, in such cases, to shorten the time, the molecule must be further modified with other molecules, requiring a great deal of effort for optimization. Thus, molecules that specifically bind to DNA mismatches, are easy to manufacture, and further exhibit characteristics such as fluorescence that facilitate the detection of DNA mismatches have not been known until now. [Means for solving the problem]

[0008] In order to solve the above-described conventional problems, the present inventors have invented a novel molecule containing an organometallic molecule that specifically binds to a specific mismatch in DNA, more specifically, a type of diaminonaphthyridine derivative having a ferrocene substituent, and a method for producing the same. This compound specifically binds to cytosine-cytosine mismatches in particular, and exhibits fluorescent and electrochemical properties. The mismatch can be detected by a simple procedure of mixing the compound with the nucleic acid to be analyzed at room temperature and measuring the fluorescence intensity using a fluorescence spectrophotometer or plate reader. Furthermore, the mismatch can be easily detected by cyclic voltammetry, which is an electrochemical measurement method, making it highly user-friendly.

[0009] The mismatch recognition molecule according to claim 1 of the present application comprises: It is a compound represented by the following chemical formula (Chemical Formula 1) that binds to cytosine-cytosine mismatches in nucleic acids.

[0010] [ka]

[0011] The mismatch detection method according to claim 2 comprises: The method is characterized by comprising the steps of: binding the mismatch recognition molecule according to claim 1 to a cytosine-cytosine mismatch in a hybridized nucleic acid; and detecting the mismatch recognition molecule bound to the mismatch by a UV spectrum measurement method or a fluorescence spectrum measurement method.

[0012] The disease diagnostic method according to claim 3 comprises: The present invention is characterized by using the mismatch recognition molecule according to claim 1 and the mismatch detection method according to claim 2.

[0013] The method for producing a mismatch recognition molecule according to claim 4 comprises: In step a, trifluoroacetic acid is added to diBoc-DAMP shown at the left end of the following chemical formula (chemical formula 2) and the mixture is stirred. In step b, ferrocenecarboxylic acid pentafluorophenyl ester produced by the following chemical formula (chemical formula 3) is added, and in step c, di-tert-butyl dioxide shown at the following chemical formula (chemical formula 4) is added and the mixture is stirred, thereby obtaining FcDAMP shown at the right end of the chemical formula (chemical formula 2) as the product.

[0014] [ka]

[0015] [ka]

[0016] [ka] [Effects of the Invention]

[0017] The mismatch recognition molecule of the present invention can specifically bind to cytosine-cytosine mismatches in a shorter time than conventional methods, without using any large-scale equipment, by simple operations that mainly involve adding the molecule to a solution of DNA to be detected and mixing and stirring, and further, cytosine-cytosine mismatches can be easily detected by its fluorescence and electrochemical properties. Specifically, when the fluorescence emitted by DNA bound to the mismatch recognition molecule of the present invention is used as an identification property, detection can be performed using not only a fluorescence spectrophotometer but also a plate reader. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a UV spectrum diagram showing the results of measuring, using an ultraviolet-visible spectrophotometer, a solution of FcDANP, which is a mismatch recognition molecule of an embodiment, and three types of solutions each comprising a mixture of FcDANP and three types of DNA having different base sequences. [Figure 2] 16 is a graph showing the results of measuring the fluorescence intensity of each of 16 types of solutions obtained by adding FcDANP, a mismatch recognition molecule of an embodiment, to 16 combinations of four types of single-stranded DNA (T2) in which the base X at a specific position is A, T, C, or G, and four types of single-stranded DNA (T17) in which the base Y at a specific position is A, T, C, or G, using a spectrofluorometer. DETAILED DESCRIPTION OF THE INVENTION

[0019] An embodiment of the present invention will be described with reference to FIGS. The mismatch recognition molecule (mismatch detection molecule) of this embodiment is a diaminonaphthyridine derivative having a ferrocene substituent that binds to a cytosine-cytosine mismatch (C-C mismatch), and this compound is referred to as FcDANP. As will be described in detail later, C-C mismatches present in nucleic acids (including both DNA and RNA) can be easily detected by measuring fluorescence intensity and the like using this FcDANP.

[0020] FcDANP, which is a mismatch recognition molecule according to this embodiment, is represented by the following chemical formula (Chemical Formula 1).

[0021] [ka]

[0022] [FcDANP manufacturing method (synthesis method)] The method for producing FcDANP (synthesis method) will be described. FcDANP, shown at the right end of (Chemical Formula 2), is produced using diBoc-DANP, shown at the left end of the following reaction formula (Chemical Formula 2), as the starting material (raw material) through steps a, b, and c as described below.

[0023] [ka]

[0024] The official name of the starting material, diBoc-DANP, is shown below. {3-[7-(3-{tert}-Butoxycarbonylamino-propylamino)-[1,8]naphthyridin-2-ylamino]-propyl}-carbamic acid tertbutylester

[0025] In step a, diBoc-DANP (19.1 mg, 0.0402 mmol) was dissolved in dry dichloromethane (2.0 mL), trifluoroacetic acid (100 mL) was added, and the mixture was stirred at room temperature for 2 hours.

[0026] In step b, the reaction solvent was evaporated under reduced pressure, and then DMF (4.0 mL), triethylamine (200 L), and 2-hydroxypyridine (380 mg, 4.00 mmol) were added to the residue in this order, and finally ferrocenecarboxylic acid pentafluorophenyl ester (15.9 mg, 0.0402 mmol) was added.

[0027] The ferrocenecarboxylic acid pentafluorophenyl ester is prepared in advance using ferrocenecarboxylic acid shown at the left end of the following reaction formula (Chemical Formula 3) as a starting material (raw material) according to the procedure described below.

[0028] [ka]

[0029] Ferrocenecarboxylic acid (207 mg, 0.900 mmol) and triethylamine (375 μL, 2.71 mmol) were mixed in THF (6.0 mL), and trifluoroacetic acid pentafluorophenyl ester (231 μL, 1.35 mmol) was added and stirred overnight at room temperature. The solution was concentrated and then purified by silica gel chromatography (n-hexane / ethyl acetate = 8 / 1) to obtain the desired product as an orange solid (339 mg, 98.5% yield).

[0030] The identification spectrum of the orange solid is shown below. 1 H NMR (400 MHz, CDCl3) δ4.98(s, 2H), 4.59(s, 2H), 4.33 (s, 5H); 13 C NMR(100MHz, CDCl3)δ168.2, 143.0, 140.5, 139.5, 136.9, 73.0, 71.2, 70.6, 67.2; HRMS(ESI)m / z calcd for C 17 H9F5FeO2(M+):395.9872, found 395.9871.

[0031] In step c, the reaction solution obtained in step b was stirred overnight at room temperature, followed by the addition of BocO (Di-tert-butyl dicarbonate (50 L)) of the following chemical formula (Chemical Formula 4), followed by further stirring at room temperature overnight. The reaction solution was diluted with ethyl acetate, and the organic layer was washed successively with water and saturated brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated, then purified by flash silica gel chromatography (methanol / ethyl acetate = 15% → 40%) and further purified by reverse-phase HPLC (10 mM aqueous ammonium acetate / acetonitrile = 1 / 1). Boc-protected FcDANP was dissolved in dry dichloromethane (2.0 mL), trifluoroacetic acid (200 L) was added, and the mixture was stirred at room temperature under nitrogen for 1.5 hours. The solvent was removed by distillation under reduced pressure to obtain FcDANP as a brown solid (7.7 mg, 32% yield).

[0032] The identification spectrum of the brown solid is shown below. HRMS(ESI) m / z calculation for C 25 H 31 FeNOM+H] + : 487.1904, found 487.1905.

[0033] [Experiment 1] Next, an experiment will be described in which CC mismatches present in DNA base sequences are detected (recognized) by fluorescence using FcDANP. First, three types of samples were prepared by adding FcDANP to DNA with a CC mismatch and two types of DNA without a CC mismatch. Additionally, FcDANP without DNA was also used as a comparison sample. Fluorescence intensity measurements were performed on these four types of samples to confirm whether CC mismatches could be detected. The details and results of the experiment are described below with reference to FIG.

[0034] A solution containing 10 μM of DNA-free FcDANP was mixed in 10 mM phosphate buffer (pH 7.0) containing 100 mM sodium chloride, and three types of solutions were obtained by mixing 50 μM of each of three types of DNA with different base sequences (CC mismatch, C-bulge, CG full match) with 10 μM of FcDANP in 10 mM phosphate buffer (pH 7.0) containing 100 mM sodium chloride. Spectra were measured using a UV-visible spectrophotometer UV1900i manufactured by Shimadzu Corporation.

[0035] FIG. 1 is a graph showing four overlapping results of measurements of the above sample taken with a UV-visible spectrophotometer, and is a UV spectrum diagram with wavelength (nm) on the horizontal axis and absorption intensity on the vertical axis.

[0036] As shown in Figure 1, the shape of the UV spectrum changes depending on the DNA sequence. However, in the wavelength range of roughly 375 nm to 400 nm, the sample containing DNA with a CC mismatch base sequence and FcDANP exhibits significantly higher absorption intensity than the other three samples, indicating that FcDANP bound to the DNA CC mismatch can be distinguished from FcDANP and samples containing DNA with other base sequences and FcDANP.

[0037] [Experiment 2] Next, an experiment was carried out in which FcDANP was added to a combination of the following two types of single-stranded DNA, T2 and T17, which were prepared by the inventors and have different base sequences, and CC mismatches were detected by measuring fluorescence intensity. T2 5'-ACA TCC AA X ACA ACC AC-3' T17 5'-GTG GTT GT Y TTG GAT GT-3' The details and results of the experiment are described below with reference to FIG.

[0038] In this experiment, four types of single-stranded DNA (T2) were prepared, each with a base X at a specific position of A, T, C, or G. Four types of single-stranded DNA (T17) were prepared, each with a base Y at a specific position of A, T, C, or G. Sixteen types of samples were prepared, each containing single-stranded DNA (T2) and single-stranded DNA (T17), with different combinations of base X in single-stranded DNA (T2) and base Y in single-stranded DNA (T17). In preparing the samples, FcDANP was 10 μM, and each DNA was 50 μM. FcDANP and DNA were mixed in 10 mM phosphate buffer (pH 7.0) containing sodium chloride.

[0039] In the experiment, the fluorescence intensity was measured at an excitation wavelength of 390 nm using a JASCO FP8300 spectrofluorometer and a four-sided transparent cell.

[0040] As shown in Figure 2, among the 16 types of samples, the only one whose measured fluorescence intensity exceeded the judgment criterion of 150 was the sample combining single-stranded DNA (T17) in which base Y was C and single-stranded DNA (T2) in which base X was C. This result shows that FcDANP specifically binds (recognizes) only the CC mismatch in DNA and emits strong fluorescence, thereby clearly distinguishing it from full-match DNA and DNA with mismatched base sequences other than the CC mismatch.

[0041] [Disease diagnostic methods using FcDANP] To determine the susceptibility of a specific individual to a disease, the FcDANP of the present invention can be used to detect a portion of the base sequence in the DNA constituting the human genome where only one base has been replaced with an unusual base (the SNP). Recent research has revealed that a CC mismatch appearing at a specific position in the DNA constituting the human genome may correspond to the onset of a specific genetic disease. Therefore, blood or the like collected from a specific individual is mixed with a reagent containing FcDANP and treated, and an experiment such as [Experiment 2] (Figure 2) is performed. If a CC mismatch is found at a specific position in the DNA, it can be determined that the specific individual is at high risk of developing that specific genetic disease.

Claims

1. A mismatch recognition molecule that binds to a cytosine-cytosine mismatch in a nucleic acid, represented by the following chemical formula (Chemical Formula 1): 【Chemical 1】

2. A mismatch detection method comprising the steps of: binding the mismatch recognition molecule according to claim 1 to a cytosine-cytosine mismatch in a hybridized nucleic acid; and detecting the mismatch recognition molecule bound to the mismatch by UV spectroscopy or fluorescence spectroscopy.

3. A disease diagnostic method using the mismatch recognition molecule of claim 1 and the mismatch detection method of claim 2.

4. A method for producing a mismatch recognition molecule, characterized in that trifluoroacetic acid is added to diBoc-DAMP shown at the left end of the following chemical formula (Chemical Formula 2) in step a, the mixture is stirred, ferrocenecarboxylic acid pentafluorophenyl ester produced by the following chemical formula (Chemical Formula 3) is added in step b, and di-tert-butyl dioxide shown in the following chemical formula (Chemical Formula 4) is further added and stirred in step c, thereby obtaining FcDAMP shown at the right end of said chemical formula (Chemical Formula 2) as a product substance. 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】

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