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

Abstract

Provided is a molecule that recognizes cytosine-cytosine mismatches in DNA. 【Solution means】Three types of solutions in which a solution of FcDANP represented by the following chemical formula is mixed with each of three types of DNAs having different base sequences and FcDANP are measured with an ultraviolet-visible spectrophotometer. In the wavelength range of approximately 375 - 400 nm, the sample containing DNA with C-C mismatch and FcDANP shows a significantly higher absorption intensity compared to others. TIFF0007712517000013.tif52170

Description

Technical Field

[0001] The present invention relates to a novel mismatch recognition molecule which is a molecule that specifically binds to a specific mismatch occurring in a nucleic acid and has a discrimination function such as fluorescence, a mismatch detection method for detecting a specific mismatch occurring in a nucleic acid by using the discrimination function of the mismatch recognition molecule, a disease diagnosis method using the mismatch recognition molecule and the mismatch detection method, and a method for producing the mismatch recognition molecule.

Background Art

[0002] When nucleic acids such as DNA and RNA hybridize to form a double strand, the combination of base pairs that pair with each other is determined. Specifically, guanine (G) pairs with cytosine (C), and adenine (A) pairs with thymine (T). Therefore, in a state where nucleic acids are hybridized, it is normal for all bases to form pairs as described above. However, in some cases, a part of the base sequence in the nucleic acid may not be able to form such a pair. Such a base pair that cannot form a normal base pair when a single-stranded nucleic acid hybridizes to form a double strand is generally called a mismatch.

[0003] In recent years, research on mismatches in the base sequence of the genome has been progressing. A typical example of a mismatch in the base sequence of the genome is a single nucleotide polymorphism (SNP) in which one base is different from the normal one due to displacement of the nucleic acid on the base sequence of the nucleic acid. According to the findings obtained in recent research, this single nucleotide polymorphism is considered to be one of the causes of genetic diseases and one of the causes of individual differences in organisms. In the field of pharmaceuticals, it is known that for drugs developed to treat a certain disease, there are patients who obtain the drug efficacy and those who do not. If these patients with different drug efficacies can be identified by SNPs, more rational and waste-free drug administration can be performed. In particular, in the case of anticancer drugs and the like, an effect of suppressing side effects can be expected.

[0004] As methods for detecting mismatches, various techniques have been proposed conventionally. In Patent Documents 1 to 3 below, inventions using compounds that form base pairs similar to mismatches are disclosed, and in Patent Documents 4 and 5 below, inventions for detecting by hybridizing another single-stranded nucleic acid are disclosed.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0006] According to the inventions disclosed in Patent Documents 1 to 3, there has been a 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 great deal of labor and expensive equipment. Further, according to the inventions disclosed in Patent Documents 4 and 5, there has been a problem that it takes time to hybridize a target nucleic acid with another single-stranded nucleic acid, and it is necessary to redesign the nucleic acid for detection depending on the base sequence near the target mismatch.

[0007] Even if a molecule that specifically binds to the type of mismatch to be detected is obtained, in order to reliably detect the molecule that specifically binds to the mismatch, it is considered preferable that the molecule has some property (special function) useful for detection, such as the property of emitting fluorescence. However, a molecule that specifically binds to a DNA mismatch and has properties useful for detection such as fluorescence has not been known conventionally, including the inventions disclosed in Patent Documents 1 to 5. This is because covalently binding a molecule synthesized aiming to impart properties such as fluorescence to a mismatch on DNA often takes a long time even using a special device depending on the base sequence of DNA, and in such cases, it is necessary to further modify the molecule with other molecules to shorten the time, etc., and a very large labor is required for optimization. Thus, a molecule that specifically binds to a DNA mismatch, is easy to manufacture, and exhibits properties such as fluorescence characteristics that facilitate the detection of DNA mismatches has not been known conventionally.

Means for Solving the Problems

[0008] In order to solve the above-described conventional problems, the inventor of the present application invented a novel molecule containing an organometal that specifically binds to a specific mismatch of DNA, more specifically, a kind of diaminonaphthyridine derivative having ferrocene as a substituent, and a method for producing the same. This compound specifically binds to a cytosine-cytosine mismatch in particular, and is a compound that exhibits fluorescence and electrochemical properties. The mismatch can be detected by a simple operation of mixing with a nucleic acid to be analyzed at room temperature and measuring the fluorescence intensity using a fluorescence spectrophotometer or a plate reader, and the mismatch can also be easily detected by cyclic voltammetry, which is one of the electrochemical measurement methods, etc., and it is excellent in usability.

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

[0010] [Chemistry]

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

[0013] Claim 3 The method for producing a mismatch recognition molecule according to is characterized in that trifluoroacetic acid is added to diBoc-DAMP shown at the left end of the following chemical formula (Chemical Formula 2) and stirred, and subsequently, the pentafluorophenyl ester of ferrocenecarboxylic acid produced by the following chemical formula (Chemical Formula 3) is added in step b, and further, di-tert-butyl dioxide shown in the following chemical formula (Chemical Formula 4) is added and stirred in step c, and FcDAMP shown at the right end of the chemical formula (Chemical Formula 2) is obtained as a product substance.

[0014] [Chemistry]

[0015] [Chemistry]

[0016] [Chemistry] [Advantages of the Invention]

[0017] The mismatch recognition molecule of the present invention can specifically bind to a cytosine-cytosine mismatch in a shorter time than before by a simple operation mainly involving adding it to a solution of the DNA to be detected and mixing and stirring without using a large-scale apparatus, and further, the cytosine-cytosine mismatch can be easily detected by its fluorescence or electrochemical properties. Specifically, when the fluorescence emitted by the DNA bound to the mismatch recognition molecule of the present invention is used as a characteristic for discrimination, detection can be performed not only with a fluorescence spectrophotometer but also with a plate reader.

Brief Description of the Drawings

[0018]

Figure 1

Figure 2

Embodiments for Carrying Out the Invention

[0019] An embodiment of the present invention will be described with reference to FIGS. 1 and 2. The mismatch recognition molecule (mismatch detection molecule) of the present embodiment is a diaminonaphthyridine derivative having ferrocene as a substituent, which binds to a cytosine-cytosine mismatch (C-C mismatch), and this compound is referred to as FcDANP. As will be described in detail later, by measuring fluorescence intensity and the like using this FcDANP, it becomes possible to easily detect C-C mismatches present in nucleic acids (including both DNA and RNA).

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

[0021] [Chemical Formula]

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

[0023] [Chemical Formula]

[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, after distilling off the reaction solvent under reduced pressure, DMF (4.0 mL), triethylamine (200 μL), 2-hydroxypyridine (380 mg, 4.00 mmol) were sequentially added to the residue, and finally ferrocenecarboxylic acid pentafluorophenyl ester (15.9 mg, 0.0402 mmol) was added.

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

[0028]

Chemical Formula

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

[0030] The identification spectrum of the orange solid is shown below. 1 H NMR (400 MHz, CDCl3) δ 4.98 (s, 2H), 4.59 (s, 2H), 4.33 (s, 5H); 13 C NMR (100 MHz, CDCl3) δ 168.2, 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, after the reaction solution obtained in step b was stirred at room temperature overnight, Boc2O (Di-tert-butyl Dicarbonate, ditert-butyl dicarbonate (50 L)) of the following chemical formula (Chemical Formula 4) was added, and the mixture was further stirred 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, concentrated, and then purified by flash silica gel chromatography (methanol / ethyl acetate = 15% → 40%), and further purified by reverse-phase HPLC (10 mM ammonium acetate aqueous solution / acetonitrile = 1 / 1). The 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 for 1.5 hours under nitrogen. The solvent was distilled off under reduced pressure to obtain FcDANP as a brown solid (7.7 mg, yield 32%).

[0032] The identification spectrum of the brown solid is shown below. HRMS(ESI) m / z calcd for C 25 H 31 FeN6O2M+H] + : 487.1904, found 487.1905.

[0033] [Experiment 1] Next, an experiment for detecting (recognizing) C-C mismatches present in the DNA base sequence by fluorescence using FcDANP will be described. First, three types of samples were prepared by adding FcDANP to DNA having a C-C mismatch and two types of DNA having no C-C mismatch, respectively. Further, FcDANP not containing DNA was also used as a comparative sample. Fluorescence intensity measurements were performed on these four types of samples to confirm whether C-C mismatches could be detected. The details and results of that experiment will be described below with reference to FIG. 1.

[0034] A solution obtained by mixing 10 μM of FcDANP without DNA in 10 mM phosphate buffer (pH 7.0) containing 100 mM sodium chloride, each of three types of 50 μM DNA with different base sequences (C-C mismatch, C-bulge, C-G full match), and 10 μM of FcDANP in 10 mM phosphate buffer (pH 7.0) containing 100 mM sodium chloride were subjected to spectral measurement using an ultraviolet-visible spectrophotometer UV1900i manufactured by Shimadzu Corporation.

[0035] Figure 1 is a graph showing the four results measured by an ultraviolet-visible spectrophotometer for the above samples superimposed, and is a UV spectrum diagram with the horizontal axis being the wavelength (nm) and the vertical axis being the absorption intensity.

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

[0037] [Experiment 2] Next, an experiment was conducted to detect C-C mismatches by adding FcDANP to a combination of the following two types of single-stranded DNA with different base sequences, T2 and T17, prepared by the inventor, and measuring the 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 that experiment will be described below with reference to Figure 2.

[0038] In this experiment, as single-stranded DNA (T2), four types with the base X at a specific position being A, T, C, and G were prepared. As single-stranded DNA (T17), four types with the base Y at a specific position being A, T, C, and G were prepared. Then, 16 types of samples containing single-stranded DNA (T2) and single-stranded DNA (T17) with different combinations of the base X of single-stranded DNA (T2) and the base Y of single-stranded DNA (T17) were prepared. In the preparation of the said samples, FcDANP was 10 μM, each DNA was 50 μM, and FcDANP and DNA were mixed in a 10 mM phosphate buffer (pH 7.0) containing sodium chloride.

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

[0040] As shown in Figure 2, among the 16 types of samples, the only sample with the measured fluorescence intensity exceeding 150 as the determination criterion was the sample combining single-stranded DNA (T17) with the base Y being C and single-stranded DNA (T2) with the base X being C. From this result, it can be seen that FcDANP can specifically bind (recognize) only to the C-C mismatch of DNA and emit strong fluorescence, thus clearly distinguishing it from fully matched DNA and DNA with a base sequence of a mismatch other than the C-C mismatch.

[0041] [Regarding the disease diagnosis method using FcDANP] In order to determine the diseases that a specific person is prone to develop, using the FcDANP of the present invention, it is possible to detect a portion (the said SNP) in which only one of the base sequences in the DNA constituting the human genome is replaced with a base different from the normal one. According to recent research, it has sometimes been found that the C-C mismatch that appears at a specific position in the DNA constituting the human genome corresponds to the expression of a specific genetic disease. Therefore, a reagent containing FcDANP is mixed with blood or the like collected from a specific person and processed, and an experiment like [Experiment 2] (Figure 2) is conducted. If a C-C mismatch is found at a specific position in the DNA, it can be determined that the specific person has a high risk of developing the 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 (Formula 1). 【Chemical 1】

2. A method for detecting a mismatch, comprising: a step of binding the mismatch recognition molecule according to Claim 1 to a cytosine-cytosine mismatch in a hybridized nucleic acid; and a step of detecting the mismatch recognition molecule bound to the mismatch by a UV spectrum measurement method or a fluorescence spectrum measurement method.

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

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