JP2009201356A - Control of formation of double-stranded dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for increasing a melting temperature (Tm) of a double-stranded DNA, and to provide a reagent for use in the method. <P>SOLUTION: An agent for increasing a melting temperature (Tm) of a DNA molecule has a G-G sequence composed of two contiguous guanine (G) residues and also has a G-G mismatch and at least one other mismatch, which comprises a naphthyridine carbamate dimer derivative represented by the general formula (1), and a method using the same. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention stabilizes a DNA molecule having a plurality of mismatches, increases the melting temperature (Tm) comprising a low molecular weight compound for increasing the melting temperature (Tm), a DNA molecule stabilizer, Alternatively, the present invention relates to a functional material having a function such as “molecular glue” for bonding unstable DNA molecules. More specifically, the present invention comprises a GG sequence in which two guanines (G) are composed of a naphthyridine carbamate dimer derivative represented by the following general formula (1), and has a GG mismatch and at least The present invention relates to a melting temperature (Tm) increasing agent for DNA molecules having another mismatch. Moreover, this invention relates to the naphthyridine carbamate dimer derivative represented by General formula (4) mentioned later. Furthermore, the present invention relates to a method for hybridizing unstable DNA molecules containing a plurality of mismatches using these low molecular compounds.

  When nucleic acids such as DNA and RNA are hybridized to form a double strand, a base to be paired is determined. For example, guanine (G) has cytosine (C) and adenine (A) has thymine (T). Usually, all the bases form such a pair and hybridize, but sometimes such a pair cannot be formed in a part of the base sequence. For example, most bases can form such a pair when subjected to conditions under which one DNA can hybridize with another DNA, but one or several partial bases May not be able to form a correct pair. Such a base pair that cannot form a normal base pair is hereinafter referred to as a mismatch.

On the other hand, studies on various genetic diseases resulting from differences in one or more bases have been conducted recently. For example, single nucleotide polymorphisms (SNPs) that are individual differences in genetic information cause susceptibility to disease and individual differences in pharmacological action. Currently, research on SNPs occupies an important position in the post-genome toward the realization of medical care optimized for each individual, and development of an efficient method for detecting SNPs is expected. DNA containing SNPs forms mismatched base pairs when mixed and annealed with complementary DNA that does not contain mutations. Therefore, if a small molecule ligand that selectively binds to the mismatched base pairs is developed, the efficiency of SNPs can be improved. It is thought that it will be possible to detect.
At present, a method for detecting such a mismatch is generally a method for comparing the hybridization efficiency of double-stranded DNA. However, in order to use this method, it is necessary to know in advance the base sequence of DNA containing a mismatch, which requires a great amount of labor, and is not suitable as a method for processing many samples. In addition, there is a technique that utilizes the selective binding of DNA repair proteins such as MutS to gene-damaged sites. However, when proteins are used, the stability and active structure ( (Folding) must be maintained, and there are many restrictions on usage conditions, and the operation is complicated, making it difficult to efficiently detect mismatches.

Thus, it is very difficult to detect some mismatches in hybridized DNA and the like, and its sensitivity is insufficient, and establishment of a method that can detect this easily and with high sensitivity is required. Yes.
By the way, the present inventors have developed a bulge DNA recognition molecule that is a molecule that specifically binds and stabilizes DNA (bulge DNA) having an unpaired base (bulge base) generated in double-stranded DNA. (See Patent Document 1). This bulge recognition molecule not only forms hydrogen bonds with unpaired bases, but also intercalates into the space created by the presence of bulge bases using the stacking interaction between the aromatic ring and bases near the bulges, It has been stabilized. The inventors of the present invention have further studied the action against unpaired bases using the stacking effect due to the presence of such surrounding bases. It has been found that a compound having two molecular species capable of forming a pair can be incorporated relatively stably by such a stacking effect. Specifically, a bis (2-methylnaphthyridine) amide derivative has a GG mismatch or a GA mismatch. It has been shown that it can be detected (see Patent Documents 2 and 3). However, this can be incorporated very sensitively and stably into a specific mismatch, but is insufficiently incorporated into other mismatches, i.e., it is too specific for a particular mismatch. It was not practical. For example, although it has high specificity for a GG mismatch, sufficient uptake has not necessarily been made for a GA mismatch or a GT mismatch.
Furthermore, the present inventors have studied many derivatives as such mismatch recognition molecules, particularly focusing on aminonaphthyridine dimers (see Patent Documents 4 to 8).

As a method for examining gene mutation, there is a method in which an oligomer DNA of about 50 bases of a gene to be examined for the presence of mutation and a wild type gene without the mutation is mixed, heated and cooled to cross-hybridize the two genes. If there is a mutation in the gene to be examined, an abnormality is found in the melting temperature or melting temperature difference of the gene. Although this operation is relatively simple, it is only known that there is a mutation, and it is impossible to know what mutation is occurring at which position.
According to the method reported by the present inventor described above (see Patent Documents 2 to 8), it is possible to know the presence of a specific mismatch by this cross-hybridization method, but this mismatch recognition molecule has specificity. For example, even when examining mismatches to guanine bases, it is necessary to prepare a plurality of mismatch recognition molecules such as a GG mismatch, a GA mismatch, and a GT mismatch.

  In addition, the present inventors have also examined in what form a mismatch recognition molecule such as aminonaphthyridine dimer is bound in the mismatch portion (see Non-Patent Document 1). For example, a naphthylidine carbamate dimer (hereinafter sometimes simply referred to as NC) binds NC: DNA in a 2: 1 stoichiometric amount in a GG mismatch sandwiched between GC base pairs. Have been reported (see Non-Patent Document 2). These analyzes have been made for one mismatch of GG, and only one mismatch in the DNA molecule does not affect the melting temperature (Tm) so much, but there are two or more mismatches. If so, the melting temperature (Tm) is greatly affected. For these reasons, attention has been paid to the behavior in mismatch for DNA molecules having a larger number of mismatches.

JP 2001-89478 A JP 2001-149096 A JP 2003-259899 A Japanese Patent Application Laid-Open No. 2004-261083 JP 2004-275179 A JP 2004-325074 A JP 2006-94725 A JP 2006-104159 A Nakatani, K., Hagihara, S., et al., Nat. Chem. Biol., 2005, 1, 39-43 Peng, T., Nakatani, K., Angew. Chem. Int. Ed. Engl., 2005, 44, 7280-7283

  The present invention provides a method for increasing the melting temperature (Tm) in double-stranded DNA, and a reagent therefor.

The present inventors have developed a mismatch recognition molecule. Such a mismatch recognition molecule recognizes a single base pair mismatch and causes a change in melting temperature (Tm). When the mismatch in the DNA molecule is 2 base pairs or more, the melting temperature (Tm) is greatly lowered, and in the extreme case, it cannot be hybridized. If such a DNA molecule can be sufficiently hybridized and the melting temperature (Tm) can be increased, not only can the DNA molecule be hybridized (adhered), but also the proximity of the molecule bound to the DNA molecule It is also possible to control withdrawal.
As a result of detailed examination of the mismatch recognition molecules developed by the present inventors, some of these mismatch recognition molecules are fused in DNA molecules having mismatches of 2 base pairs or more. It has been found that the molecule can extremely raise the temperature (Tm).
That is, the present invention provides the following general formula (1)

(In the formula, each of R ¹ and R ² is independently a hydrogen atom, a hydrocarbon group having 1 to 5 carbon atoms, or one or more carbon atoms of the hydrocarbon group having 1 to 5 carbon atoms, R represents a group substituted with an atom selected from the group consisting of oxygen atoms, R represents a linear or branched alkylene group having 1 to 10 carbon atoms, X represents an oxygen atom or a sulfur atom, R ³ Is a hydrogen atom or the following general formula (2)

(In the formula, each of R ¹ and R ² is independently a hydrogen atom, a hydrocarbon group having 1 to 5 carbon atoms, or one or more carbon atoms of the hydrocarbon group having 1 to 5 carbon atoms, Represents a group substituted with an atom selected from the group consisting of oxygen atoms, R represents a linear or branched alkylene group having 1 to 10 carbon atoms, X represents an oxygen atom or a sulfur atom, and L represents Indicates a linker group that connects two nitrogen atoms.)
The group represented by these is shown. )
The melting temperature (Tm) of a DNA molecule having two guanines (G) having a continuous GG sequence and having a GG mismatch and at least one other mismatch, consisting of a naphthyridine carbamate dimer derivative represented by It relates to the agent.
Further, the present invention provides a melting temperature (Tm) increasing agent for a DNA molecule comprising the naphthyridine carbamate dimer derivative represented by the general formula (1), and has a GG sequence in which two guanines (G) are continuous. And a method for increasing the melting temperature (Tm) of a DNA molecule in the solution by adding the DNA molecule having a GG mismatch and at least another mismatch to the solution.
Furthermore, the present invention relates to a solution in which a DNA molecule having a GG sequence in which two guanines (G) are continuous and having a GG mismatch and at least another mismatch is present, the melting temperature ( Tm) At a temperature equal to or higher than that, the melting temperature (Tm) of the DNA molecule comprising the naphthyridine carbamate dimer derivative represented by the general formula (1) is added to increase the melting temperature of the DNA molecule in the solution (Tm). The present invention relates to a method of increasing the Tm) and hybridizing the DNA molecule.
Further, the present invention provides the following general formula (4)

(In the formula, each of R ¹ and R ² independently represents a hydrogen atom, a hydrocarbon group having 1 to 5 carbon atoms, or one or more carbon atoms of the hydrocarbon group having 1 to 5 carbon atoms, Represents a group substituted by an atom selected from the group consisting of oxygen atoms, R represents a linear or branched alkylene group having 1 to 10 carbon atoms, X represents an oxygen atom or a sulfur atom, and L represents The following general formula (3)
—R ⁴ —Ar ¹ —N═N—Ar ² —R ⁵ — (3)
(Wherein R ⁴ and R ⁵ each independently represent a linear or branched alkylene group having 1 to 5 carbon atoms, Ar ¹ and Ar ² each independently represent an arylene group, and an azo group Is thin or anti-configuration.)
The group represented by these is shown. )
It is related with the novel naphthyridine carbamate dimer derivative represented by these.
Furthermore, the present invention provides an agent for increasing the melting temperature (Tm) of a DNA molecule comprising a naphthyridine carbamate dimer derivative represented by the general formula (4), and has a GG sequence in which two guanines (G) are continuous. And increasing the melting temperature (Tm) of the DNA molecule in the solution by adding it to a solution in which a DNA molecule having a GG mismatch and at least one other mismatch is present, and two guanines A solution in which (G) has a continuous GG sequence and a DNA molecule having a GG mismatch and at least another mismatch is present at a temperature equal to or higher than the melting temperature (Tm) of the DNA molecule. Adding a melting temperature (Tm) increasing agent for a DNA molecule comprising a naphthyridine carbamate dimer derivative represented by the formula (4) More raising the melting temperature (Tm) of the DNA molecules in the solution, to a method for hybridizing the DNA molecule. More specifically, the above-described method relates to the above-described method, which is based on syn-anti isomerization of an azo group by irradiation with light of the naphthyridine carbamate dimer derivative represented by the general formula (4).

The present invention will be described more specifically as follows.
(1) The following general formula (1)

(In the formula, each of R ¹ and R ² is independently a hydrogen atom, a hydrocarbon group having 1 to 5 carbon atoms, or one or more carbon atoms of the hydrocarbon group having 1 to 5 carbon atoms, R represents a group substituted with an atom selected from the group consisting of oxygen atoms, R represents a linear or branched alkylene group having 1 to 10 carbon atoms, X represents an oxygen atom or a sulfur atom, R ³ Is a hydrogen atom or the following general formula (2)

(In the formula, each of R ¹ and R ² is independently a hydrogen atom, a hydrocarbon group having 1 to 5 carbon atoms, or one or more carbon atoms of the hydrocarbon group having 1 to 5 carbon atoms, Represents a group substituted with an atom selected from the group consisting of oxygen atoms, R represents a linear or branched alkylene group having 1 to 10 carbon atoms, X represents an oxygen atom or a sulfur atom, and L represents Indicates a linker group that connects two nitrogen atoms.)
The group represented by these is shown. )
The melting temperature (Tm) of a DNA molecule having two guanine (G) having a continuous GG sequence and having a GG mismatch and at least one other mismatch, comprising a naphthyridine carbamate dimer derivative represented by Agent.
(2) The elevating agent according to (1), wherein an increase in melting temperature (Tm) when measured under the same conditions is at least 15 ° C. or higher.
(3) The elevating agent according to (1) or (2), wherein the increase in melting temperature (Tm) when measured under the same conditions is at least 25 ° C. or higher.
(4) The elevating agent according to any one of (1) to (3), wherein the DNA molecule is a DNA molecule having another mismatch at a position adjacent to the GG mismatch.
(5) The elevating agent according to (4), wherein the other mismatch is a TG mismatch at a position adjacent to the GG mismatch.
(6) The elevating agent according to any one of (1) to (4), wherein the DNA molecule has three or more mismatches.
(7) The elevating agent according to (6), wherein the third mismatch is at a position adjacent to the GG mismatch.
(8) The above (1) to (7), wherein the mismatch in the DNA molecule is 5′-TGG-3 ′ / 3′-GGT-5 ′ or 5′-TGG-3 ′ / 3′-GGC-5 ′. The raising agent in any one of.
(9) The increase according to any one of (1) to (8), wherein R ¹ and R ² in the general formulas (1) and (2) are each independently an alkyl group having 1 to 5 carbon atoms. Agent.
(10) The elevating agent according to (9), wherein R ¹ and R ² in the general formulas (1) and (2) are methyl groups.
(11) The elevating agent according to any one of (1) to (10), wherein X in the general formulas (1) and (2) is an oxygen atom.
(12) The elevating agent according to any one of (1) to (11), wherein R in the general formulas (1) and (2) is a propylene group.
(13) The elevating agent according to any one of (1) to (12), wherein the linker group L in the general formula (2) contains an azo group.
(14) The linker group L in the general formula (2) is represented by the following general formula (3)
—R ⁴ —Ar ¹ —N═N—Ar ² —R ⁵ — (3)
(Wherein R ⁴ and R ⁵ each independently represent a linear or branched alkylene group having 1 to 5 carbon atoms, Ar ¹ and Ar ² each independently represent an arylene group, and an azo group Is thin or anti-configuration.)
The raising agent as described in said (13) which is group represented by these.
(15) The elevating agent according to (14), wherein R ⁴ and R ⁵ in the general formula (3) are methylene groups, and Ar ¹ and Ar ² are p-phenylene groups.
(16) The elevating agent according to (14) or (15), wherein the azo group in the general formula (3) is in a syn configuration.

(17) A GG sequence in which two guanines (G) are continuously used as a melting temperature (Tm) elevating agent for a DNA molecule comprising the naphthyridine carbamate dimer derivative according to any one of (1) to (16) And a melting point (Tm) of the DNA molecule in the solution is increased by adding to a solution in which a DNA molecule having a GG mismatch and at least one other mismatch is present.
(18) A solution in which two guanine (G) has a continuous GG sequence and a DNA molecule having a GG mismatch and at least one other mismatch is not lower than the melting temperature (Tm) of the DNA molecule. The melting temperature (Tm) of the DNA molecule comprising the naphthyridine carbamate dimer derivative according to any one of (1) to (16) above is added at a temperature of 5 to melt the DNA molecule in the solution. A method of increasing the temperature (Tm) to hybridize the DNA molecule.
(19) The following general formula (4)

(In the formula, each of R ¹ and R ² independently represents a hydrogen atom, a hydrocarbon group having 1 to 5 carbon atoms, or one or more carbon atoms of the hydrocarbon group having 1 to 5 carbon atoms, Represents a group substituted by an atom selected from the group consisting of oxygen atoms, R represents a linear or branched alkylene group having 1 to 10 carbon atoms, X represents an oxygen atom or a sulfur atom, and L represents The following general formula (3)
—R ⁴ —Ar ¹ —N═N—Ar ² —R ⁵ — (3)
(Wherein R ⁴ and R ⁵ each independently represent a linear or branched alkylene group having 1 to 5 carbon atoms, Ar ¹ and Ar ² each independently represent an arylene group, and an azo group Is thin or anti-configuration.)
The group represented by these is shown. )
A naphthyridine carbamate dimer derivative represented by:
(20) The naphthyridine carbamate dimer derivative according to (19), wherein R ¹ and R ² in General Formula (4) are each independently an alkyl group having 1 to 5 carbon atoms.
(21) The naphthyridine carbamate dimer derivative according to (20), wherein R ¹ and R ² in the general formula (4) are methyl groups.
(22) The naphthyridine carbamate dimer derivative according to any one of (19) to (21), wherein X in the general formula (4) is an oxygen atom.
(23) The naphthyridine carbamate dimer derivative according to any one of (19) to (22), wherein R in the general formula (4) is a propylene group.
(24) The naphthyridine carbamate dimer derivative according to any one of (19) to (23), wherein R ⁴ and R ⁵ in the general formula (3) are methylene groups, and Ar ¹ and Ar ² are p-phenylene groups. .
(25) The melting temperature (Tm) elevating agent for a DNA molecule comprising the naphthyridine carbamate dimer derivative according to any one of (19) to (24), wherein the guanine (G) is a continuous GG sequence. And adding to a solution in which a DNA molecule having a GG mismatch and at least another mismatch is present, thereby increasing the melting temperature (Tm) of the DNA molecule in the solution.
(26) A solution in which two guanine (G) has a continuous GG sequence and a DNA molecule having a GG mismatch and at least one other mismatch is not lower than the melting temperature (Tm) of the DNA molecule. The melting temperature of the DNA molecule in the solution (Tm) by increasing the melting temperature (Tm) of the DNA molecule comprising the naphthyridine carbamate dimer derivative according to any one of (19) to (24) Tm) is raised to hybridize the DNA molecule.
(27) The method according to (25) or (26), wherein the method according to (25) or (26) is based on syn-anti isomerization of an azo group by light irradiation.
(28) The following general formula (10),
^{Z 1 -N (Z 2) -R} 4 -Ar 1 -N = N-Ar 2 -R 5 -N (Z 3) -Z 4 (10)
(Wherein R ⁴ and R ⁵ each independently represent a linear or branched alkylene group having 1 to 5 carbon atoms, Ar ¹ and Ar ² each independently represent an arylene group, and an azo group Is a thin or anti configuration, and Z ¹ and Z ⁴ are represented by the following general formula (11)

(In the formula, R ⁸ is selected from the group consisting of a hydrogen atom, a hydrocarbon group having 1 to 5 carbon atoms, or one or more carbon atoms of the hydrocarbon group having 1 to 5 carbon atoms consisting of a nitrogen atom and an oxygen atom) R represents a linear or branched alkylene group having 1 to 10 carbon atoms, and X represents an oxygen atom or a sulfur atom.
Z ² and Z ³ each independently represent a hydrogen atom or a group represented by the general formula (11). )
A naphthyridine carbamate derivative having an azo group represented by:
(29) The naphthyridine carbamate derivative according to the above (28), wherein Z ² and Z ³ in the general formula (11) are hydrogen atoms.
(30) The following equation (9)

An azobenzene derivative represented by
(31) DNA molecule stabilization for reversibly switching the stability of an unstable DNA molecule containing a mismatch comprising the compound according to any one of (28) to (30) by photoisomerization by light irradiation Agent.
(32) The stability of an unstable DNA molecule containing a mismatch comprising any one of the compounds described in (28) to (30) above, reversibly by photoisomerization by light irradiation. Melting temperature (Tm) control agent. More specifically, an increasing or decreasing agent for the melting temperature (Tm) of the DNA molecule.

The present inventors have analyzed the structure of a naphthyridine carbamate dimer derivative in a single-base mismatch having a sequence of 5′-CGG-3 ′ / 3′-GGC-5 ′ (see Non-Patent Documents 1 and 2). From these results, the present inventors have found that the naphthyridine carbamate dimer derivative, particularly, R ¹ and R ² in the general formula (1) are methyl groups, R is a propylene group, and R ³ is hydrogen. A naphthyridine carbamate dimer derivative (hereinafter sometimes abbreviated as NC), which is an atom and X is an oxygen atom, forms a hydrogen bond with guanine (G), and two molecules of NC form a continuous sequence of GG It was assumed that hydrogen bonds. This assumption will be described with reference to FIG. FIG. 1a (left side of FIG. 1) shows the state of hydrogen bonding between NC and guanine (G), and FIG. 1b (right side of FIG. 1) shows NC for a single-base mismatch sequence of GG. This is a schematic illustration of how to enter. Two molecules of NC hydrogen bond to a continuous GG sequence. As a result, it was assumed that the base adjacent to the continuous GG sequence was put out of the DNA helix. Then, the present inventors considered that if this assumption is correct, the base adjacent to the continuous GG sequence need not be a matching cytosine (C), and even a mismatched base may be possible. . In FIG. 1b, this base is indicated by X. In order to prove this hypothesis, the present inventors then experimented with a two-base pair mismatch having the sequence 5′-TGG-3 ′ / 3′-GGC-5 ′.

For this purpose, DNA consisting of 11 bases (T1 / C1) was prepared. Each base sequence is
T1: 5′-CCCA TG GTCCG-3 ′
C1: 3′-GGGT GG CAGGC-5 ′
And has two mismatches of TG and GG (see underlined portion).
The melting temperature (Tm) of this DNA (T1 / C1) (5 μM) in 10 mM sodium cacodylate (pH = 7.0) buffer containing 0.1 M NaCl was 35.7 ° C. NC 100 μM was added thereto, and the melting temperature (Tm) was measured in the same manner. As a result, it was 71.1 ° C. Indeed, an increase in melting temperature of 35.7 ° C. could be observed. The result is shown in FIG. The horizontal axis of FIG. 2 indicates temperature (° C.), and the vertical axis indicates absorbance at 260 nm. The temperature was increased at a rate of 1 ° C. per minute, and the absorbance was measured 3 times every 1 ° C., and the average value is shown in the graph of FIG. The upper curve (black in the original drawing) in FIG. 2 shows a case where NC is not present, and the lower curve (red in the original drawing) shows a case where NC is present.
As can be seen from FIG. 2, particularly in the range of 45 ° C. to 55 ° C., when NC is not present, T1 and C1 are present as a single chain, but when NC is present, T1 and C1 exist in a double strand.

In order to further confirm this, a CSI-TOF (cold spray ionization timing-of-flight) mass spectrum was taken. FIG. 3 shows a range of 1000-1800 as a result of CSI-TOF mass spectrum in the presence or absence of NC 40 μM in a 50% aqueous methanol solution containing 20 mM of T1 and C1 and 100 mM ammonium acetate. The sample was cooled to −10 ° C. and injected at a rate of 0.5 mL / hour. 3a (left side of FIG. 3) is in the absence of NC, and FIG. 3b (right side of FIG. 3) is in the presence of NC.
As shown in FIG. 3a, single-stranded T1 was observed as a 3 ^- ion ([T1] ³⁻ ), and m / z was observed at 1096.2 (calculated value: 1096.2). Moha double stranded T1 and C1 5 ^- as an ion ^{([T1 / C1] 5-)} , m / z was observed at 1344.8 (calculated value: 1344.6). On the other hand, by adding NC, a peak of 2: 1 complex ([T1 / C1 + 2NC] ⁵⁻ ) with NC, which is a new peak, was observed at m / z 1546.3 (calculated value: 1545). .9).
In addition, when the concentration of NC was changed, it was observed that the single-stranded T1 ion ([T1] ³⁻ ) decreased and the complex with NC ([T1 / C1 + 2NC] ⁵⁻ ) increased. It was. 4a (left side of FIG. 4) is a CSI-TOF mass spectrum when the concentration of NC is 20 μM (T1: NC = 1: 1) with respect to 20 μM T1, and FIG. 4b (right side of FIG. 4) is T1 of 20 μM. It is a CSI-TOF mass spectrum in case the density | concentration of NC with respect to is 60 micromol (T1: NC = 1: 3). As a result, it was shown that the single-stranded T1 ion ([T1] ³⁻ ) decreased and the complex with NC ([T1 / C1 + 2NC] ⁵⁻ ) increased with increasing NC concentration. .

In this case, the present inventors predicted that thymine (T) in the 5′-TGG-3 ′ / 3′-GGC-5 ′ sequence would be outside the DNA helix. In order to confirm this, the present inventors tried to oxidize DNA (T1 / C1) in which double strands were formed by NC with potassium permanganate. If thymine (T) is outside the helix, it is oxidized by potassium permanganate to thymine glycol (Tg) (Hayatsu, H .; Ukita, T., Biochem. Biophys. Res. Comm., 1967, 29, 556-561). Oxidized thymine glycol (Tg) is then cleaved at this site by heating in piperidine (Rubin, CM; Schmid, CW, Nucleic. Acids Res., 1980, 8, 4613-20; Maxam, AM; Gilbert, W., Methods Enzymol., 1980, 65, 499-560; Gogos, JA; Karayiorgou, M., et al., Nucleic. Acids Res., 1990, 18, 6807-6814).
Double stranded DNA (T1 / C1) was oxidized with potassium permanganate at 0 ° C. and then treated in piperidine at 90 ° C. The outline of this reaction is shown in the following scheme.

Double-stranded T1 / C1 in the absence of NC did not react for 320 minutes with 0.2 mM potassium permanganate, but double-stranded T1 / C1 in the presence of 40 μM NC. Then, Tg1 (T1 chain oxidized to thymine glycol) was confirmed by HPLC. The presence of fragments cleaved by heat treatment in piperidine was confirmed by HPLC. These HPLC charts are shown in FIG. FIG. 5a (upper left of FIG. 5) shows an HPLC of the result of treatment with potassium permanganate in the absence of NC, and FIG. 5b (upper right of FIG. 5) shows with potassium permanganate in the presence of NC. Fig. 5c (lower left side of Fig. 5) shows the HPLC of the result of heat treatment with piperidine after treatment with potassium permanganate in the presence of NC. As a result, the peak of Tg1 could be confirmed in FIG. 5b, and the fragments CCCAp and pGGTCCG cleaved by heat treatment in piperidine could be confirmed.
These fragments were confirmed by MALDI-TOF mass spectrum. T1 before oxidation is observed at m / z 3293.5 (calculated value: 3293.2), and T1 of oxidized thymine glycol (Tg), that is, Tg1 is observed at m / z 3326.1. (Calculated value: 3327.2).
And m / z of the fragment 5′-d (CCCA) -PO ₃ H-3 ′ (CCCAp) obtained by heat-treating this in piperidine was observed at 118.6 (calculated value: 198.8). ), M / z of the other fragment 5′-HO ₃ Pd (GGTCCG) -3 ′ (pGGTCCG) was observed at 1888.4 (calculated value: 1888.2).
These oligomers were also dephosphorylated at the end with alkaline phosphatase and confirmed in reverse phase HPLC along with standard oligomers.
Two thymines (T) exist in T1 consisting of 11 bases. That is, thymine (T) in -ATGG- and thymine (T) in -GGTCC- exist. However, in the treatment with potassium permanganate and subsequent treatment with piperidine, only the former, ie, thymine (T) in -ATGG- was reacted. This result proves that only the thymine (T) is present outside the DNA helix due to the presence of NC.

From these results, it is proved that NC is a reagent that can increase the melting temperature (Tm) of DNA containing two mismatches and can exist stably as double-stranded DNA. The mismatch base other than guanine has a structure that is driven out of the DNA helix.
In addition, we have further confirmed that these results are universal in that a 3 base pair of 5′-TGG-3 ′ / 3′-GGT-5 ′ sequence is used. Experiments were performed on mismatches, ie, TG mismatch, GG mismatch, and GT mismatch.
For this purpose, DNA consisting of 11 bases (T2 / T3) was prepared. Each base sequence is
T2: 5′-CCTT TGG TCAG-3 ′
T3: 3′-GGAA GGT AGTC-5 ′
And has three mismatches of TG, GG, and GT (see the underlined portion). This DNA (T2 / T3) exists as a single-stranded DNA at room temperature, but becomes a double-stranded DNA as the NC concentration increases. The melting temperature (Tm) in the presence of 100 μM NC reached 58.8 ° C. Thus, the melting temperature (Tm) of this DNA (T2 / T3) (5 μM) in a 10 mM sodium cacodylate (pH = 7.0) buffer containing 0.1 M NaCl was below room temperature. However, when 100 μM of NC was added thereto and the melting temperature (Tm) was measured in the same manner, it was 58.8 ° C. Indeed, an increase in melting temperature of 30 ° C. or higher could be observed. Although the melting temperature (Tm) of DNA (T2 / T3) could be measured up to 10 ° C. or less, it was difficult to accurately measure the melting temperature (Tm).
The heat denaturation characteristics of this DNA (T2 / T3) (5 μM) are shown in FIG. The horizontal axis in FIG. 6 represents temperature (° C.), and the vertical axis represents absorbance at 260 nm. The temperature was increased at a rate of 1 ° C. per minute, and the absorbance was measured three times every 1 ° C., and the average value is shown in the graph of FIG. The eight curves in FIG. 6 show the cases where the concentration of NC is 0, 2.5, 5, 10, 20, 40, 60, and 100 μM from the bottom of the right end (around 80 ° C.), respectively.
The transition from single strand to double strand of T2 and T3 at each concentration of NC was observed by CD spectrum. NC concentrations of 0, 5, 10, 15, and 20 μM at 25 ° C. in 10 mM sodium cacodylate (pH = 7.0) buffer containing 5 μM each of T2 and T3 and 0.1 M NaCl. Each CD spectrum at was measured. The results are shown in FIG. (A) in FIG. 7 shows a case where NC is 0, that is, non-existence, (b) shows a case where NC is 5 μM, (c) shows a case where NC is 10 μM, (d) shows a case where NC is The case of 15 μM is shown, and (e) shows the case where NC is 20 μM. As a result, when NC was not present, a positive band was shown at 272 nm and a negative band was shown at 250 nm. However, as the molar ratio was increased from 1 to 4 by addition of NC, a positive band was obtained. It was shown to increase elliptically. In addition, the intensity of the CD bands at 348 nm and 324 nm changed with increasing concentration of NC. This CD change includes a point of isodichroic, indicating a transition from a single strand of T2 and T3 to a double strand containing NC.

The formation of a double-stranded complex containing T2 and T3 NCs was also confirmed by CSI-TOF mass spectrum. Absence of NC in a 50% aqueous methanol solution containing 20 mM of T2 and T3 containing 100 mM ammonium acetate (FIG. 8 (a)), NC was present at 20 μM (FIG. 8 (b)), and NC was present at 40 μM ( FIG. 8 shows a range of 1000 to 1800 as a result of CSI-TOF mass spectrum when (c) in FIG. 8) and NC is present at 60 μM ((d) in FIG. 8). The sample was cooled to −10 ° C. and injected at a rate of 0.5 mL / hour. 8 (a) (upper left side of FIG. 8) is in the absence of NC, and FIG. 8 (b) (upper right side of FIG. 8) is in the presence of 20 μM of NC, FIG. 8 (c). (Lower left side in FIG. 8) is in the presence of 40 μM NC, and FIG. 8D (lower right side in FIG. 8) is in the presence of 60 μM NC.
As shown in FIG. 8A, single-stranded T2 was observed as 3 ^- ion ([T2] ³⁻ ), and m / z was observed at 1107.6 (calculated value: 106.2). Single-stranded T3 was also observed as 3 ^- ion ([T3] ³⁻ ), and m / z was observed at 1140.2 (calculated value: 1138.9). The T2 and that of the two strands of T3 5 ^- as an ion ^{([T2 / T3] 5-)} , m / z was observed at 1349.0 (calculated value: 1347.2). In addition, it was a double-stranded one of T2 and T3 and was observed as 4 ^- ion ([T2 / T3] ^4- ), and m / z was observed at 1687.2 (calculated value: 1684.3). On the other hand, when NC was added in an amount of 20 μM, that is, a molar ratio of 1: 1 ((b) in FIG. 8), a 2: 1 complex ([T2 / T3 + 2NC]) with the new NC was obtained. The peak of ^5- ) was observed at m / z 1550.7 (calculated value: 1548.5).
Further, when the concentration of NC is changed, single-stranded T2 ions ([T2] ³⁻ ) and T3 ions ([T3] ³⁻ ) are decreased, and a complex with NC ([T2 / T3 + 2NC] ⁵⁻ ) Increased (see FIG. 8C (lower left side in FIG. 8) and FIG. 8D) (lower right side in FIG. 8)).
Even in this case, formation of a complex with a stoichiometric amount of 1: 2 molar ratio of DNA: NC was confirmed.

Further, in the same manner as described above, oxidative cleavage with potassium permanganate was conducted using DNA consisting of the following 11 bases.
T4: 5′-GCAA TGG TTGC-3 ′
T4: 3′-CGTT GGT AACG-5 ′
This reaction scheme is shown next as in the above scheme.

Treatment of T4 in the absence of NC with 0.2 mM potassium permanganate at 0 ° C. for 40 minutes did not react (see FIG. 9 (a)), but 2 in the presence of 40 μM NC. In the main chain T4 / T4, products 1 and 2 were confirmed after the subsequent heat treatment with pyrimidine (see FIG. 9B). By treating with 0.2 mM potassium permanganate for 5 hours, the products 1 and 2 can be obtained as main products (see FIG. 9C). Tg4 (T4 chain oxidized to thymine glycol) obtained by treatment with 0.2 mM potassium permanganate appears in the immediate vicinity of T4 in HPLC and was difficult to confirm by HPLC. The products 1 and 2 were obtained by the subsequent heat treatment of piperidine, which could be easily confirmed. The products 1 and 2 obtained here were confirmed by MALDI-TOF mass spectrum. That is, product 1 is oligomer 5′-d (GCAA) —PO ₃ H-3 ′, m / z is observed at 1261.6 (calculated value: 1262.2), and product 2 is oligomer 5′- HO ₃ Pd (GGTTGC) -3 ′, m / z was observed at 1901.7 (calculated value: 1902.3). In the presence of alkaline phosphatase, these oligomers were dephosphorylated by treatment at 37 ° C. for 30 minutes to give 5′-d (GCAA) -3 ′ and 5′-d (GGTTGC) -3 ′, respectively. This was compared with that of the standard product and confirmed to have the same retention time (see FIG. 9 (d)).
These HPLC charts are shown in FIG. FIG. 9 (a) (upper left of FIG. 9) shows the HPLC of the result of treatment with potassium permanganate in the absence of NC, and FIG. 9 (b) (upper right of FIG. 9) shows the presence of 40 μM NC. 9 shows HPLC after treatment with potassium permanganate for 40 minutes, followed by heat treatment in piperidine, FIG. 9 (c) (bottom left of FIG. 9) shows permanganate in the presence of 40 μM NC. Shown is HPLC after 5 hours treatment with potassium followed by heat treatment in piperidine. FIG. 9 (d) (lower right side of FIG. 9) shows the HPLC results after treating products 1 and 2 with alkaline phosphatase.
As a result, the peak of Tg1 could be confirmed in FIG. 5b, and the fragments CCCAp and pGGTCCG cleaved by heat treatment in piperidine could be confirmed.
Further, the MALDI-TOF mass spectrum of T4 before oxidation was observed at m / z of 3372.01 (calculated value: 3370.60), and T4 of oxidized thymine glycol (Tg), that is, Tg4 was m / z. z was observed at 3404.37 (calculated value: 3404.61). From this, the stoichiometric structure similar to the 2 base pair mismatch having the 5′-TGG-3 ′ / 3′-GGC-5 ′ sequence described above is 5′-TGG-3 ′ / 3. It was confirmed that even a mismatch of 3 base pairs having the sequence of “-GGT-5” occurred.
From these results, it is possible to increase the melting temperature (Tm) and stabilize as a double-stranded DNA for DNA molecules having 3 mismatches, as in the case of DNA containing 2 mismatches as described above. Proved to be a reagent that can be present in It was proved that the mismatch bases other than guanine had a structure that was driven out of the DNA helix.

These experimental results demonstrate that the naphthyridine carbamate dimer derivative (NC) exhibits a sufficient stabilizing effect in DNA having two or more mismatches, that is, an effect of increasing the melting temperature (Tm). Is. This is because such a naphthyridine carbamate dimer derivative (NC) can stabilize and hybridize single-stranded DNAs that are different in base sequence to such an extent that they cannot hybridize in a normal temperature range. It demonstrates that it has the property of being able to. And this fact is not limited to pyrimidine bases such as cytosine (C) and thymine (T), but also in purine bases such as adenine (A) and guanine (G), that is, due to the presence of NC. It also indicates that the base of the mismatched base pair is driven out of the DNA helix.
As described above, the naphthyridine carbamate dimer derivatives (NCs) disclosed in the present invention have not only the phenomenon of stabilization of DNA and an increase in melting temperature (Tm), but also the structure of DNA by π- It has been clarified specifically by the present invention that it has an action of stabilizing the structure by the stacking effect.

The naphthyridine carbamate dimer derivative (NCs) of the present invention is represented by the general formula (1) or the naphthyridine carbamate dimer derivative represented by the general formula (1) will be described in more detail.
In the general formula (1) of the present invention, R ¹ and R ² are each independently one or more carbon atoms of a hydrogen atom, a hydrocarbon group having 1 to 5 carbon atoms, or a hydrocarbon group having 1 to 5 carbon atoms. Is a group substituted with an atom selected from the group consisting of a nitrogen atom and an oxygen atom. Here, the hydrocarbon group having 1 to 5 carbon atoms may be saturated or unsaturated, and may be linear or branched. Examples of such hydrocarbon groups include linear or branched alkyl groups having 1 to 5 carbon atoms, linear or branched alkenyl groups having 2 to 5 carbon atoms, and cycloalkyl groups having 3 to 5 carbon atoms. Can be mentioned. Specific examples of such hydrocarbon groups include alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and pentyl groups; Examples thereof include alkenyl groups such as vinyl group, allyl group, isopropenyl group, butenyl group and pentenyl group; alkynyl groups such as ethynyl group and propynyl group. Preferable hydrocarbon groups include linear or branched alkyl groups having 1 to 5 carbon atoms such as methyl group, ethyl group, propyl group, and isopropyl group. In addition, as the group in which at least one carbon atom of the hydrocarbon group having 1 to 5 carbon atoms in R ¹ and R ² is substituted with an atom selected from the group consisting of a nitrogen atom and an oxygen atom, A C 1-4 alkoxyl group in which the carbon atom is substituted with an oxygen atom, a C 1-4 alkoxyalkyl group, a C 1-4 dialkylamino group in which the carbon atom is substituted with a nitrogen atom, C1-C4 (di or mono) -alkylaminoalkyl groups, C1-C3 alkoxyalkoxy groups in which two carbon atoms are substituted with oxygen atoms, and the like are limited thereto. is not.

X in the general formula (1) of the present invention includes an oxygen atom or a sulfur atom. When X is an oxygen atom, a carbamate group is formed with an adjacent amide group, and when X is a sulfur atom, a thiocarbamate group is formed with an adjacent amide group. Thus, the carbamate group in the present invention may be a carbamate group composed of the oxygen source s, or a thiocarbamate group in which one oxygen atom is substituted with a sulfur atom. Preferred carbamate groups include carbamate groups consisting of oxygen atoms, that is, carbamate groups where X is an oxygen atom.
R in the general formula (1) of the present invention represents a linear or branched alkylene group having 1 to 10 carbon atoms, and specifically includes, for example, a methylene group, an ethylene group, a propylene group, and a butylene group. Linear alkylene group, methylmethylene group, ethylmethylene group, 1-methylethylene group, 2-methylethylene group, 1-ethylethylene group, 2-ethylethylene group, 1-methylpropylene group, 2-methylpropylene group And branched alkylene groups such as 3-methylpropylene group, 1-ethylpropylene group, 2-ethylpropylene group, and 3-ethylpropylene group. Preferable R is an alkylene group in which two naphthyridine rings form a hydrogen bond with guanine or the like, have a length and a degree of freedom to obtain a sterically π-stacking effect, and have excellent thermal stability of the molecule. Specific examples thereof include a straight-chain propylene group having 3 carbon atoms or an alkyl group-substituted product thereof. More preferable R includes a linear propylene group.

R ^{3 in} the general formula (1) may be a hydrogen atom when the naphthyridine carbamate dimer represented by the general formula (1) of the present invention is used alone, but is not limited thereto. Absent. Examples of the group other than a hydrogen atom include a group having 1 to 5 carbon atoms, preferably about 1 to 3 carbon atoms, and a chemically inert group such as an alkyl group.
In addition, the naphthyridine carbamate dimer represented by the general formula (1) of the present invention has this dimer structure because it binds to a DNA molecule in a stoichiometric amount of 1: 2 as described above. It can also be used as a thing. As those having such a dimeric structure, include those having a structure in which R ³ in the general formula (1) is represented by the general formula (2). R ¹ , R ² , R, and X in the general formula (2) are the same as described above. L in the general formula (2) is not particularly limited as long as each naphthyridine carbamate dimer moiety can be bonded by a chemical bond, and the distance and the degree of freedom of both can be appropriately maintained. L is preferably a linear or branched alkylene group having 5 to 15 carbon atoms, an alkylene group having a phenylene group having 12 to 20 carbon atoms, the following general formula (3)
—R ⁴ —Ar ¹ —N═N—Ar ² —R ⁵ — (3)
(Wherein R ⁴ and R ⁵ each independently represent a linear or branched alkylene group having 1 to 5 carbon atoms, Ar ¹ and Ar ² each independently represent an arylene group, and an azo group Is thin or anti-configuration.)
And a group containing an azo group represented by:
Examples of R ⁴ and R ⁵ in the general formula (3) include a linear or branched alkylene group having 1 to 5 carbon atoms, such as a methylene group, an ethylene group, a propylene group, and a butylene group. A preferable alkylene group includes a methylene group. Examples of the arylene group represented by Ar ¹ and Ar ² include a divalent arylene group derived from an aryl group composed of a 6-membered aromatic ring having 6 to 30 carbon atoms, preferably 6 to 12 carbon atoms. Preferable arylene groups include phenylene groups such as p-phenylene group, m-phenylene group and o-phenylene group. Preferable Ar ¹ and Ar ² include a p-phenylene group. The azo group in the general formula (3) may be in a syn configuration or an anti configuration, but it will be described later that the stability of the DNA molecule varies depending on the configuration of the azo group.
As a preferable compound in General formula (1) of this invention, following formula (5)

A naphthyridine in which R ¹ and R ² in the general formula (1) are a methyl group, R is a propylene group, R ³ is a hydrogen atom, and X is an oxygen atom Carbamate dimer derivative, the following formula (6) or (7)

In other words, R ¹ and R ² in the general formula (1) are methyl groups, R is a propylene group, and R ³ is N- (azo-bis (p-phenylenemethylene)). -Anti-isomer (6) and syn isomer (7) among naphthyridine carbamate dimers, which are naphthyridine carbamate dimers and X is an oxygen atom.

Of the naphthyridine carbamate dimer derivatives represented by the general formula (1) of the present invention, the compound in which R ³ is a hydrogen atom is a known compound, and can be produced by, for example, the method described in Patent Document 8. . For example, N, N-bis (hydroxyalkyl) amine or an N-protected form thereof is carbonate protected with an O-protecting group such as a succinoyl group, and this includes 2-amino-1,8-naphthyridine or its 7-position. It can be produced by reacting 2-amino-1,8-naphthyridine substituted with R ¹ or R ² . As the protecting group in this case, an amino protecting group used in peptide synthesis such as hydrochloride, acyl group or alkoxycarbonyl group can be used.

The compound in which R ³ in the general formula (1) of the present invention is other than a hydrogen atom, that is, the compound represented by the general formula (4) is a novel compound.
Examples of the compound represented by the general formula (4) of the present invention include the following general formula (8).
OHC—R ⁶ —Ar ¹ —N═N—Ar ² —R ⁷ —CHO
(In the formula, Ar ¹ and Ar ² each independently represent an arylene group, and R ⁶ and R ⁷ each independently represent a chemical bond or a linear or branched alkylene group having 1 to 4 carbon atoms. )
Can be produced by reacting the compound in which R ³ in the general formula (1) of the present invention is a hydrogen atom under reducing conditions to couple an amino group. As these reaction conditions, the usual conditions for reductive amination reaction can be employed.

The compound represented by the general formula (4) of the present invention recognizes a base such as naphthyridine in the molecule, that is, a site capable of hydrogen bonding with the base, and causes syn-anti isomerization by light irradiation. It has the azo group which can be formed.
For example, by irradiating the anti isomer (6) (trans isomer) with light of 360 nm, the syn isomer (7) (cis isomer) can be obtained. By irradiation with light of 430 nm, it can be reversibly converted to the anti form (6) (trans form). The reaction formulas for these photoisomerizations are shown below.

In this way, the syn isomer and the anti isomer that are reversibly converted by irradiation with light have a structure that is significantly different sterically, and the interaction with the base is sterically limited. This situation is schematically shown in FIG. Molecules having such an azo group are reversibly converted by light 1 and light 2 to take a cis-trans three-dimensional structure. For example, as shown in FIG. 10, when it becomes a syn isomer (cis isomer), it can interact with mismatched DNA in the same manner as NC described above, and can stabilize a DNA molecule having mismatch. However, even if it is in a state stabilized by such a three-dimensional structure, when light 2 is irradiated here and changed to an anti isomer (trans isomer), it no longer has a stable interaction. The steric structure cannot be taken and the DNA molecule becomes unstable.
For example, double strand DNA 5′-CTA ACG GAA TG-3 ′ / 3′-GAT TGG CTT AC-5 ′ containing GG mismatch and trans-form-3 represented by formula (6) are mixed and The melting temperature (Tm) of the strand DNA was measured. 360 nm light irradiation is carried out for 5 minutes to convert from trans-form-3 (i) to cis-form-3 (ii). At this time, the melting temperature (Tm) of the double-stranded DNA molecule significantly increases from 29 ° C. to 52 ° C. (see FIG. 11), and takes a stable double-stranded structure. Furthermore, when 430 nm light is irradiated to cis-form-3 (ii), it will return to trans-form-3 (iii). Then again the duplex is greatly destabilized due to the presence of mismatched base pairs. Further irradiation with 360 nm light returns the cis-form-3 (iv) again. The results of measuring the heat denaturation characteristics in each of these states are shown in FIG. The horizontal axis in FIG. 11 indicates temperature (° C.), and the vertical axis indicates absorbance (AU). In FIG. 11, (i) shows the case of trans isomer-3 (i), (ii) shows the case of cis isomer-3 (ii), and (iii) shows the case of trans isomer-3 (iii). (Iv) shows the case of cis-form-3 (iv), respectively.
In this way, the function of “molecular glue” can be revived by light irradiation. As described above, it was possible to reversibly control DNA double strand formation with small molecules and light.

As described above, the naphthyridine carbamate dimer derivative represented by the general formula (1) of the present invention increases and stabilizes the melting temperature (Tm) of a DNA molecule having two or more mismatches. This is because the naphthyridine carbamate dimer derivative represented by the general formula (1) of the present invention binds to the GG site in the mismatch sequence to induce and stabilize a double-stranded structure containing the mismatch. . That is, the naphthyridine carbamate dimer derivative represented by the general formula (1) of the present invention exhibits a function as a “molecular glue” for converting unstable DNA having a mismatch from a single strand to a double strand. Because it does. The naphthyridine carbamate dimer derivative having an azo group represented by the general formula (4) of the present invention functions as such a “molecular glue”, and further, DNA double-strand formation is reversible by an external stimulus such as light. It can be said that the device has a function for controlling it. A molecule in which such an azobenzene skeleton having photoisomerization ability is introduced into the linker portion of the naphthyridine carbamate dimer derivative of the present invention is a compound represented by the general formula (4) of the present invention. The trans / cis of the azobenzene moiety is converted by light irradiation, and the binding ability of the trans isomer and cis isomer to the mismatched DNA also changes accordingly (see FIG. 10). That is, the compound of the general formula (4) can control the on / off switching of the function as the “molecular glue” depending on the wavelength of the light to be irradiated.
Examples of the compound that can be controlled by light include not only the naphthyridine carbamate dimer derivative represented by the general formula (4) of the present invention described above, but also a simple chemical structure represented by the following formula: (9)

The compound represented by these is mentioned. This compound has a chemical structure in which one naphthyridine structure is deleted from the nitrogen atom, and the on / off switching of the function as the “molecular glue” described above can be controlled with a simple structure.
Therefore, when the compound that can control the on / off switching of the function as “molecular glue” according to the wavelength of light to be irradiated in the present invention is represented by the following general formula (10),
^{Z 1 -N (Z 2) -R} 4 -Ar 1 -N = N-Ar 2 -R 5 -N (Z 3) -Z 4 (10)
(Wherein R ⁴ and R ⁵ each independently represent a linear or branched alkylene group having 1 to 5 carbon atoms, Ar ¹ and Ar ² each independently represent an arylene group, and an azo group Is a thin or anti configuration, and Z ¹ and Z ⁴ are represented by the following general formula (11)

(In the formula, R ⁸ is selected from the group consisting of a hydrogen atom, a hydrocarbon group having 1 to 5 carbon atoms, or one or more carbon atoms of the hydrocarbon group having 1 to 5 carbon atoms consisting of a nitrogen atom and an oxygen atom) R represents a linear or branched alkylene group having 1 to 10 carbon atoms, and X represents an oxygen atom or a sulfur atom.
Z ² and Z ³ each independently represent a hydrogen atom or a group represented by the general formula (11). )
It can be called a naphthyridine carbamate derivative having an azo group represented by the formula:
R ⁴ , R ⁵ , Ar ¹ , and Ar ² in the general formula (10) of the present invention are the same as those described above, and R and X in the general formula (11) are also as described above. the same meanings, ^{R 8} in the general formula (11) represents the same group as ^{R 1} and ^{R 2} in the general formula (1) that has been said.
A naphthyridine carbamate derivative having an azo group represented by the general formula (10) is also a novel compound and can be produced by the same method as the compound represented by the general formula (4).

Next, the DNA molecule in the present invention will be described. In the present invention, “a DNA molecule having two guanine (G) having a continuous GG sequence and having a GG mismatch and at least one other mismatch” means (1) at least two G as a base sequence. Has a continuous GG sequence, (2) has a GG mismatch, and (3) has at least one mismatch in addition to the above-mentioned GG mismatch. Furthermore, as a preferable embodiment, there is a GG mismatch shown in the above (2) in one guanine (G) in a GG sequence in which the two guanine (G) shown in the above (1) is continuous, Other mismatches shown in 3) are adjacent to the GG mismatch shown in (2). That is, as a preferred embodiment, a DNA molecule having a base sequence of 5′-NGG-3 ′ / 3′-GGY-5 ′ (N and Y each independently represents a base other than cytosine (C)). Is mentioned. More preferred embodiments include DNA molecules having a base sequence in which at least one of N or Y, preferably both are thymine (T). A preferred mismatch other than the GG mismatch in the present invention includes a GT mismatch.
The “mismatch” in the present invention is a base other than the Watson-Crick base pair, that is, the TA or GC base pair, and also includes a bulge base. Therefore, although the case where one or both of N or Y in the above-mentioned sequence is not a base is included, the case where N and Y are bases other than cytosine (C) is mentioned as a preferred embodiment.
The length of the DNA molecule of the present invention is not particularly limited, but if it is too long, the base portion that matches the mismatched portion increases and the melting temperature (Tm) does not decrease. 6 to 150 mer, more preferably 10 to 50 mer, still more preferably about 10 to 30 mer. When there are many mismatched portions, the length is not limited to the above-described length, and may be longer. Preferable DNA molecules include DNA molecules having mismatches such that double-stranded DNA cannot be formed in the range of 0 ° C. to 40 ° C., preferably 10 ° C. to 30 ° C.

The melting temperature (Tm) in the present invention is a meaning used for ordinary nucleic acids. That is, when a nucleic acid, such as DNA, is heated, an extreme change occurs in the three-dimensional structure at a certain temperature, and this change is equivalent to a phase transition. (Tm). For example, when measuring the UV absorption of a DNA solution having a double helix structure in a thin neutral salt solution while raising the temperature, the temperature is almost constant until reaching a certain temperature. When the UV absorption increased rapidly and reached a value where the UV absorption increased by about 40 to 50%, it became constant again (dark color effect). Since the temperature range of this change is extremely narrow and it looks as if a phase transition has occurred, the temperature in the middle of the temperature range at which the change has occurred as DNA has been “melted” is called the melting temperature (Tm). In the present invention, “melting temperature (Tm)” is used in this sense. The melting temperature can be measured not only by ultraviolet absorption but also by circular dichroism and infrared absorption.
In the present invention, “increase in melting temperature (Tm)” means a naphthyridine carbamate dimer derivative represented by general formula (1), a naphthyridine carbamate dimer derivative represented by general formula (4), or a general formula ( 11) Under the same measurement conditions as those measured in the absence of any of the naphthyridine carbamate derivatives represented by 11), preferably in the presence of any of the aforementioned naphthyridine carbamate dimer derivatives, the DNA molecules and these compounds When comparing the melting temperature (Tm) when measured in the presence of a molar ratio (ratio of equivalents in the case of the general formula (4)) of 1: 1 or more, more preferably 1: 2 or more, When these compounds of the present invention are present, it means an increase of at least 15 ° C., preferably 25 ° C. or more. Therefore, there are no particular restrictions on the measurement conditions of the “melting temperature (Tm)” in the present invention, for example, the type of solvent, the type of salt, the concentration of salt, and the concentration of DNA, but extreme concentrations and selection of solvents are excluded. It is natural to be done. There is no particular limitation as long as the measurement is performed with a thin neutral salt solution.

The “DNA molecule melting temperature (Tm) elevating agent” of the present invention stabilizes a mismatched DNA molecule by the presence of the naphthyridine carbamate dimer derivative of the present invention and increases the melting temperature (Tm). Speaking of the same temperature, it means that a DNA molecule that can exist only in a single strand can be stabilized into a double-stranded DNA, and the “melting temperature (Tm) of the DNA molecule of the present invention”. The “elevating agent” has a function of “glue” that makes a DNA molecule existing in a single-stranded state into a double strand, that is, “adheres” such a DNA molecule. In this sense, the “DNA molecule melting temperature (Tm) increasing agent” of the present invention can be referred to as a “molecular glue” that bonds two molecules together, or a stabilizer of double-stranded DNA. It can also be called a double-stranded DNA forming agent at a constant temperature.
Furthermore, as described above, the naphthyridine carbamate dimer derivative represented by the general formula (4) and the naphthyridine carbamate derivative represented by the general formula (11) of the present invention are reversibly synthesized by irradiation with light. Since it contains an azo group that can be isomerized, the function as the “molecular glue” described above can be turned on or off by light irradiation. Therefore, a molecule capable of turning on or off the function as the “molecular glue” of the present invention increases or decreases the melting temperature (Tm) of the DNA molecule, that is, at room temperature. Since the DNA double strand can be stabilized or made unstable at a certain constant temperature, it can be referred to as a melting temperature (Tm) control agent for the DNA molecule. It can also be referred to as an increasing agent and / or decreasing agent for the melting temperature (Tm) of the DNA molecule.

The “melting temperature (Tm) increasing agent of DNA molecule” of the present invention has not only a function of stabilizing unstable DNA having a mismatch but also a function of “molecular glue” as described above. Therefore, various applications are possible.
For example, two types of substances (eg, fluorescent dye, organic molecule, nucleic acid molecule, protein, sugar, vesicle, metal particle, metal thin film, cell, vesicle, etc.) are labeled on each strand of the DNA molecule of the present invention having a mismatch. Then, these two kinds of substances can be brought close to each other only in the presence of the “DNA molecule melting temperature (Tm) increasing agent” of the present invention. Moreover, if the raising agent of this invention which has an azo group is used, it will become possible to make these two types of reversible substances spatially close or away by irradiation with light.
In addition, it is known that the assembly of heterologous or homologous proteins is important in the intracellular signal transduction system, and the function of the “molecular glue” of the present invention is used only in the presence of the elevating agent of the present invention. It becomes possible to assemble two proteins. Using this method, it is also possible to examine the interaction between heterologous proteins. Furthermore, if a molecular glue that switches with light is used, a signal transmission system based on protein-protein interaction can be easily turned on / off by light irradiation.
Furthermore, by immobilizing the elevating agent of the present invention on the substrate, two kinds of substances can be spatially brought close to each other only on the spot where the elevating agent of the present invention is immobilized, and fluorescence emission or fluorescence It becomes possible to measure the interaction of two kinds of substances such as quenching of light by a simple method. By using this method, it is possible to easily determine the presence of any two substances.

As described above, the present invention provides a novel reagent capable of spatially approaching or moving two kinds of molecules at the molecular level and a method using the same based on a completely new concept. .
In many conventional methods for controlling the structure of nucleic acids, it is necessary to introduce a labeling substance into DNA in advance by modifying DNA at a site that responds to light or the like. For this reason, such a method has no effect on natural DNA, and its use has been greatly limited. On the other hand, in the present invention, since the structure of the nucleic acid is controlled by adding a small molecule from the outside, there is no need to modify the DNA itself. That is, it can target long-chain DNA and intracellular DNA that are difficult to synthesize. In addition, it is possible to control the structure reversibly and only to a specific arrangement by light irradiation. The present invention is based on the unique idea that nucleic acid molecules with many mismatches can be stabilized by the addition of small molecules, and the method of the present invention can be extended to a wide variety of mismatch molecules. The present invention provides the basic idea.
Further, according to the present invention, DNA bonding can be controlled by small molecules and light. For example, hybridization on a DNA chip, control of nanostructures based on nucleic acids, various biochemical phenomena associated with nucleic acid duplex formation, and control by small molecules such as replication and transcription are possible.

EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.
In addition, the compound represented by Formula (5) used in the following examples was produced by the method described in Patent Document 8.

Measurement of heat denaturation properties of DNA molecules (T1 / C1).
A DNA molecule (T1 / C1) consisting of 11 bases having the following base sequence was produced.
T1: 5′-CCCA TG GTCCG-3 ′
C1: 3′-GGGT GG CAGGC-5 ′
5 μM of this DNA molecule (T1 / C1) was measured for thermal denaturation characteristics in a range of 4 ° C. to 94 ° C. in a 10 mM sodium cacodylate (pH = 7.0) buffer containing 0.1 M NaCl. . The temperature was increased at a rate of 1 ° C. per minute, the absorbance at 260 nm was measured 3 times every 1 ° C., and the average value was measured. The result is shown by the upper curve in FIG.
Similarly, 5 μM of this DNA molecule (T1 / C1) and 100 μM of a naphthyridine carbamate dimer derivative (hereinafter simply referred to as NC) represented by the formula (5) were added to 10 mM sodium cacodylate containing 0.1 M NaCl. The heat denaturation property in the range of 4 ° C. to 94 ° C. was measured in a buffer (pH = 7.0).
The result is shown by the lower curve in FIG.
Moreover, these melting temperatures (Tm) were measured. As a result, the melting temperature (Tm) of the DNA molecule (T1 / C1) in the absence of NC was 35.7 ° C. NC 100 μM was added thereto, and the melting temperature (Tm) was measured in the same manner. As a result, it was 71.1 ° C.
As a result, it was found that the NC of the present invention increases the abduction temperature (Tm) of the DNA molecule.

Measurement of CSI-TOF (cold spray ionization timt-of-flight) mass spectrum of DNA molecule (T1 / C1).
20 μM of each of DNA molecules T1 and C1 prepared in Example 1 was dissolved in a 50% aqueous methanol solution containing 100 mM ammonium acetate, cooled to −10 ° C., and CSI-TOF at a rate of 0.5 mL / hour. And injected into the measurement. The results for m / z in the range 1000-1800 are shown in FIG.
Similarly, in the presence of 20 μM of each of the DNA molecules T1 and C1 and 40 μM of NC, the range of m / z of 1000-1800 as a result of CSI-TOF mass spectrum is shown in FIG. 3b.
Single ^- stranded T1 ([T1] ³⁻ ); measured value 1096.2
Calculated 1099.9.
Double-stranded T1 / C1 ([T1 / C1] ⁵⁻ ); measured value 1344.8
Calculated value 1344.6.
2: 1 complex with NC ([T1 / C1 + 2NC] ⁵⁻ ); found 1546.3
Calculated 1545.9.

Oxidation of DNA molecule (T1 / C1) followed by cleavage by piperidine heating.
12.5 μM of each of the DNA molecules T1 and C1 prepared in Example 1 was dissolved in a 10 mM sodium cacodylate (pH = 7.0) buffer containing 100 mM sodium chloride. 2 mM was added and reacted at 0 ° C. for 320 minutes. The HPLC of the resulting product is shown in FIG. As a result, no reaction occurred.
Similarly, 12.5 μM of each of DNA molecules T1 and C1 and 40 μM of NC are dissolved in a buffer of 10 mM sodium cacodylate (pH = 7.0) containing 100 mM sodium chloride. 2 mM was added and reacted at 0 ° C. for 320 minutes. The HPLC of the resulting product is shown in FIG. As a result, generation of Tg1 (T1 chain oxidized to thymine glycol) was confirmed.
The isolated Tg1 was heat treated with piperidine at 90 ° C. for 30 minutes. The resulting HPLC is shown in FIG.

Measurement of heat denaturation properties of DNA (T2 / T3).
A DNA molecule (T2 / T3) consisting of 11 bases having the following base sequence was produced.
T2: 5′-CCTT TGG TCAG-3 ′
T3: 3′-GGAA GGT AGTC-5 ′
The heat denaturation characteristics of 5 μM of this DNA molecule (T2 / T3) are the same as in Example 1 for each of the cases where the NC concentration is 0, 2.5, 5, 10, 20, 40, 60, and 100 μM. Measured. The results are shown in FIG. The eight curves in FIG. 6 show the cases where the concentration of NC is 0, 2.5, 5, 10, 20, 40, 60, and 100 μM from the bottom of the right end (around 80 ° C.), respectively.
Moreover, the transition from single strand to double strand of T2 and T3 at each concentration of NC was observed by CD spectrum. NC concentrations of 0, 5, 10, 15, and 20 μM at 25 ° C. in 10 mM sodium cacodylate (pH = 7.0) buffer containing 5 μM each of T2 and T3 and 0.1 M NaCl. Each CD spectrum at was measured. The results are shown in FIG.
Moreover, it was 58.8 degreeC when the melting temperature (Tm) when 100 micromol NC was added was measured.

Measurement of CSI-TOF mass spectrum of DNA (T2 / T3).
20 μM each of the DNA molecules T2 and T3 prepared in Example 4 was dissolved in a 50% aqueous methanol solution containing 100 mM ammonium acetate, cooled to −10 ° C., and converted to CSI-TOF at a rate of 0.5 mL / hour. Injected. The results are shown in FIG.
Similarly, the CSI-TOF mass when NC is present at 20 μM (FIG. 8B), NC is present at 40 μM (FIG. 8C), and NC is present at 60 μM (FIG. 8D). The spectrum was measured. The resulting range of 1000-1800 is shown in FIG.
Single ^- stranded T2 ([T2] ³⁻ ); measured value 1107.6
Calculated 1106.2.
Single ^- stranded T3 ([T3] ³⁻ );
Calculated value 1138.9.
Double ^- stranded T2 / T3 ([T2 / T3] ⁵⁻ ); measured value 1349.0
Calculated value 1347.2.
Double ^- stranded T2 / T3 ([T2 / T3] ⁴⁻ ); found 1687.2
Calculated value 1684.3.
2: 1 complex with NC ([T2 / T3 + 2NC] ⁵⁻ ); found 1550.7
Calculated value 1548.5).

Oxidation of DNA (T4 / T4) followed by cleavage by piperidine heating.
A DNA molecule (T4 / T4) consisting of 11 bases having the base sequence shown below was produced.
T4: 5′-GCAA TGG TTGC-3 ′
T4: 3′-CGTT GGT AACG-5 ′
Treatment of T4 in the absence of NC with 0.2 mM potassium permanganate at 0 ° C. for 40 minutes did not react (see FIG. 9 (a)), but 2 in the presence of 40 μM NC. In the main chain T4 / T4, products 1 and 2 were confirmed after the subsequent heat treatment with pyrimidine (see FIG. 9B). By treating with 0.2 mM potassium permanganate for 5 hours, products 1 and 2 could be obtained as main products (see FIG. 9 (c)).
Tg4 (T4 chain oxidized to thymine glycol) was difficult to confirm by HPLC, but it can be easily confirmed because products 1 and 2 are obtained by heat treatment of piperidine following oxidation. It was. The products 1 and 2 obtained here were confirmed by MALDI-TOF mass spectrum.
Product 1: oligomer _{5'-d (GCAA) -PO 3} H-3 '
Actual value 1261.6
Calculated 1262.2.
Product 2: Oligomer 5′-HO ₃ Pd (GGTTGC) -3 ′
Actual value 1901.7
Calculated value 1902.3.
Moreover, the MALDI-TOF mass spectrum of T4 and Tg4 before being oxidized was measured. T4: measured value 3372.01
Calculated value 3370.60.
Tg4; measured value 3404.37
Calculated value 3404.61.

Production of azobenzene derivative represented by formula (9) An azobenzene derivative represented by formula (9) was produced according to the following reaction formula.

4,4′-azobenzaldehyde (Compound No. 11 in the above reaction formula) (79.7 mg, 0.335 mmol) and a naphthyridine derivative (183 mg, 0.703 mmol) represented by Compound No. 12 in the above reaction formula Is added to a mixed solvent of chloroform (10 mL), methanol (5 mL), and acetic acid (50 mL), and the mixture is stirred at room temperature for 15 minutes. A solution of sodium cyanotrihydroborate (43 mg, 0.684 mmol) in methanol (1 mL) is added dropwise thereto. After stirring at room temperature for 2 hours, a saturated aqueous sodium bicarbonate solution is added and the mixture is extracted with chloroform, and the organic phase is washed with saturated brine. The organic phase was concentrated and collected under reduced pressure, and then purified by silica gel column chromatography (chloroform: methanol = 100: 5 to 100: 20) to give the title compound (Compound No. 1 in the above reaction formula) (90.6 mg). 0.125 mmol) was obtained with a yield of 37%.
¹ H NMR (CDCl ₃ , 400 MHz) δ
8.25 (d, J = 8.8 Hz, 2H), 8.11 (d, J = 9.0 Hz, 2H),
7.98 (d, J = 8.3 Hz, 2H), 7.85 (d, J = 8.3 Hz, 4H, Azobenzene),
7.65 (brs, 2H, NH), 7.47 (d, J = 8.3 Hz, 4H, Azobenzene), 7.25 (d, 2H),
4.35 (t, J = 6.3 Hz, 4H, CH2), 3.90 (s, 4H, Bn),
2.80 (t, J = 6.6 Hz, 4H, CH2), 2.76 (s, 6H, CH3),
1.95 (pseudo quintet, 4H, CH2);
ESIMS calculated value: [M + Na] ⁺ 749.33,
Actual value: 749.28.

Production of azobenzene dimer derivative represented by formula (6) An azobenzene dimer derivative represented by formula (6) was produced according to the following reaction formula.

(1) 4,4′-azobenzaldehyde (Compound No. 11 in the above reaction formula) (26.2 mg, 0.110 mmol) and naphthyridine carbamate dimer (62. 3 mg, 0.124 mmol) is added to a mixed solvent of chloroform (1.5 mL), acetonitrile (4.5 mL), and acetic acid (7 mL), and the mixture is stirred at room temperature for 2 hours. To this, sodium cyanotrihydroborate (6.9 mg, 0.110 mmol) is added and stirred at room temperature for 4 days. Saturated aqueous sodium hydrogen carbonate is added, extracted with chloroform, and the organic phase is washed with saturated brine. The organic phase was concentrated and collected under reduced pressure, and then purified by silica gel column chromatography (chloroform: methanol = 100: 2 to 100: 2.5) to obtain a monoalkylamino represented by compound number 14 in the above reaction formula. The aldehyde (36.9 mg, 0.051 mmol) was obtained in 46% yield.
¹ H NMR (CDCl ₃ , 400 MHz) δ
10.06 (s, 1H, CHO), 8.26 (d, J = 8.8 Hz, 2H), 8.06 (d, J = 8.8 Hz, 2H),
7.89 (d, J = 8.3 Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H),
7.86 (d, J = 7.3 Hz, 2H), 7.77 (d, J = 8.3 Hz, 2H), 7.60 (brs, 2H, NH),
7.50 (d, J = 8.3 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H),
4.31 (t, J = 6.2 Hz, 4H, CH2), 3.64 (s, 2H, Bn), 2.72 (s, 6H, CH3),
2.61 (t, J = 6.6 Hz, 4H, CH2), 1.92 (pseudo quintet, 4H, CH2);
ESIMS calculated value: [M + Na] ⁺ 748.30,
Actual value: 748.14.

(2) Monoalkylaminoaldehyde 14 (32.8 mg, 0.045 mmol) obtained in (1) above and naphthyridine carbamate dimer (32.4 mg, 0.064 mmol) represented by compound number 13 in the above reaction formula ) In a mixed solvent of chloroform (2.5 mL), acetonitrile (2 mL), and acetic acid (6.2 mL), and the mixture is stirred at 40 ° C. for 2 hours. To this, sodium cyanotrihydroborate (4.2 mg, 0.067 mmol) is added and stirred at 35 ° C. for 12 hours. Saturated aqueous sodium hydrogen carbonate is added, extracted with chloroform, and the organic phase is washed with saturated brine. The organic phase was concentrated and collected under reduced pressure, and then purified by silica gel column chromatography (chloroform: methanol: acetic acid = 100: 5: 0.5 to 100: 8: 0.5) to give the title compound (the above reaction formula Compound No. 2) (17.8 mg, 0.015 mmol) was obtained in a yield of 33%.
¹ H NMR (CDCl ₃ , 400 MHz) δ
8.27 (d, J = 9.0 Hz, 4H), 8.09 (d, J = 8.8 Hz, 4H),
7.92 (d, J = 8.1 Hz, 4H), 7.69 (d, J = 8.6 Hz, 4H, Azobenzene),
7.68 (brs, 4H, NH), 7.39 (d, J = 8.3 Hz, 4H, Azobenzene),
7.22 (d, J = 8.3 Hz, 4H), 4.32 (t, J = 6.5 Hz, 8H, CH2),
3.62 (s, 4H, Bn), 2.73 (s, 12H, CH3), 2.61 (t, J = 6.6 Hz, 8H, CH2),
1.93 (pseudo quintet, 4H, CH2);
ESIMS calculated value: [M + Na] ⁺ 1235.53,
Actual measurement value: 1234.93.

Photoisomerization of the azobenzene dimer derivative represented by the formula (6) produced in Example 8.
4.5 mM of double-stranded DNA 5′-CTA ACG GAA TG-3 ′ / 3′-GAT TGG CTT AC-5 ′ containing GG mismatch, and photosensitivity represented by the formula (6) produced in Example 8 Sex ligand 2 (6.8 mM) was mixed and the melting temperature (Tm) of the double-stranded DNA was measured. 360 nm light irradiation is performed for 5 minutes to convert from a trans form to a cis form. At this time, the melting temperature (Tm) of the double-stranded DNA molecule significantly increases from 29 ° C. to 52 ° C. (see FIG. 11), and takes a stable double-stranded structure. Furthermore, when 430 nm light is irradiated to the cis form (ii), it returns to the trans form 3 (iii). Then again the duplex is greatly destabilized due to the presence of mismatched base pairs. Furthermore, when 360 nm light irradiation is performed, the function of “molecular glue” can be revived. As described above, it was possible to reversibly control DNA double strand formation with small molecules and light.

The present invention relates to a melting temperature (Tm) increasing agent for stabilizing a DNA molecule that has become unstable due to mismatching by the addition of a low molecular weight compound, a method using the same, and a novel method therefor It provides chemical substances.
The reagent and method of the present invention are useful not only in various assays such as nucleic acids such as DNA and proteins, but also in identification and analysis of functions of nucleic acids and proteins. It has industrial applicability as a chemical for diagnostic agents.

FIG. 1a (left side of FIG. 1) shows the state of hydrogen bonding between a naphthyridine carbamate dimer derivative (NC) and guanine (G), and FIG. 1b (right side of FIG. 1) shows one base of GG. This schematically shows how NC enters the mismatch sequence. FIG. 2 shows the thermal denaturation of DNA (T1 / C1) (5 μM) solution in the presence of the naphthyridine carbamate dimer derivative (NC) of the present invention (lower curve in FIG. 2) and in the absence (upper side of FIG. 2). It is a graph which shows the result measured by (curve). FIG. 3 shows the results of CSI-TOF mass spectra in the presence (FIG. 3b) or absence (FIG. 3a) of the naphthyridine carbamate dimer derivative (NC) of the present invention in a 20 μM solution of DNA molecules T1 and C1. The range of 1000-1800 is shown. FIG. 4 shows the CSI-TOF mass spectrum in the presence of 20 μM of the naphthyridine carbamate dimer derivative (NC) of the present invention (FIG. 4a) or in the presence of 60 μM (FIG. 4b) in a 20 μM solution of DNA molecules T1 and C1. The range of the result 1000-1800 is shown. FIG. 5 shows permanganese in the presence (FIG. 5b) or absence (FIG. 5a) of the naphthyridine carbamate dimer derivative (NC) of the present invention of double-stranded DNA (T1 / C1) in the absence of NC. It is a HPLC chart of the result of the oxidation reaction with potassium acid. FIG. 5c (lower left side of FIG. 5) shows the HPLC of the result of heat treatment with piperidine after treatment with potassium permanganate in the presence of NC. FIG. 6 shows the heat denaturation properties of DNA (T2 / T3) (5 μM). The horizontal axis in FIG. 6 represents temperature (° C.), and the vertical axis represents absorbance at 260 nm. The eight curves in FIG. 6 indicate that the concentration of the naphthyridine carbamate dimer derivative (NC) of the present invention is 0, 2.5, 5, 10, 20, 40, 60, and 100 μM from below the right end (around 80 ° C.). Each case is shown. FIG. 7 shows the results of measuring the transition from single strand to double strand of T2 and T3 at various concentrations of the naphthyridine carbamate dimer derivative (NC) of the present invention by CD spectrum. (A) in FIG. 7 shows a case where NC is 0, that is, non-existence, (b) shows a case where NC is 5 μM, (c) shows a case where NC is 10 μM, (d) shows a case where NC is The case of 15 μM is shown, and (e) shows the case where NC is 20 μM. FIG. 8 shows the result of confirming the formation of a double-stranded complex containing the naphthyridine carbamate dimer derivative (NC) of T2 and T3 of the present invention by CSI-TOF mass spectrum. Absence of NC (FIG. 8 (a)), NC 20 μM present (FIG. 8 (b)), NC 40 μM present (FIG. 8 (c)), and NC 60 μM present (FIG. 8 (d) )) Shows the range of 1000-1800 as a result of CSI-TOF mass spectrum. FIG. 9 shows the oxidation reaction of DNA molecule T4 with potassium permanganate in the absence (FIG. 9 (a)) or presence (FIG. 9 (b)) of the naphthyridine carbamate dimer derivative (NC) of the present invention, It is the HPLC chart of the product after the heat processing with a subsequent pyrimidine. FIG. 9 (c) (bottom left of FIG. 9) shows the HPLC after 5 hours treatment with potassium permanganate in the presence of 40 μM NC followed by heat treatment in piperidine. FIG. 9 (d) (lower right side of FIG. 9) shows the HPLC results after treating products 1 and 2 with alkaline phosphatase. FIG. 10 schematically shows that the syn isomer and the anti isomer, which are reversibly converted by light irradiation, have three-dimensionally different structures, and the interaction with the base is sterically restricted. It is shown. FIG. 11 shows the results of measuring the thermal denaturation characteristics of DNA molecules by light irradiation using the azobenzene dimer derivative represented by the formula (6) of the present invention. In FIG. 11, (i) shows the case of trans-form-3 (i), (ii) shows the case of cis-form-3 (ii), and (iii) shows the case of trans-form-3 (iii). (Iv) shows the case of cis-form-3 (iv), respectively.

T1: 5′-CCCATGGTCCG-3 ′
T2: 5′-CCTTTGGTCAG-3 ′
T4: 5′-GCAATGGTTGC-3 ′

Claims (27)

The following general formula (1)

(In the formula, each of R ¹ and R ² is independently a hydrogen atom, a hydrocarbon group having 1 to 5 carbon atoms, or one or more carbon atoms of the hydrocarbon group having 1 to 5 carbon atoms, R represents a group substituted with an atom selected from the group consisting of oxygen atoms, R represents a linear or branched alkylene group having 1 to 10 carbon atoms, X represents an oxygen atom or a sulfur atom, R ³ Is a hydrogen atom or the following general formula (2)

(In the formula, each of R ¹ and R ² is independently a hydrogen atom, a hydrocarbon group having 1 to 5 carbon atoms, or one or more carbon atoms of the hydrocarbon group having 1 to 5 carbon atoms, Represents a group substituted with an atom selected from the group consisting of oxygen atoms, R represents a linear or branched alkylene group having 1 to 10 carbon atoms, X represents an oxygen atom or a sulfur atom, and L represents Indicates a linker group that connects two nitrogen atoms.)
The group represented by these is shown. )
The melting temperature (Tm) of a DNA molecule having two guanine (G) having a continuous GG sequence and having a GG mismatch and at least one other mismatch, comprising a naphthyridine carbamate dimer derivative represented by Agent.   The raising agent according to claim 1 whose rise in melting temperature (Tm) when measured on the same conditions is at least 15 ° C or more.   The raising agent of Claim 1 or 2 whose raise of a melting temperature (Tm) when measured on the same conditions is at least 25 degreeC or more.   The elevating agent according to any one of claims 1 to 3, wherein the DNA molecule is a DNA molecule having another mismatch at a position adjacent to the GG mismatch.   The raising agent according to claim 4, wherein another mismatch is a TG mismatch at a position adjacent to the GG mismatch.   The raising agent according to any one of claims 1 to 4, wherein the DNA molecule has three or more mismatches.   The elevating agent according to claim 6, wherein the third mismatch is at a position adjacent to the GG mismatch.   The mismatch in the DNA molecule is 5'-TGG-3 '/ 3'-GGT-5' or 5'-TGG-3 '/ 3'-GGC-5'. Elevating agent. Increasing agent according to any one of claims 1 to 8 R ¹ and R ² are each independently an alkyl group of 1 to 5 carbon atoms in the general formula (1) and (2). The increasing agent according to claim 9, wherein R ¹ and R ² in the general formulas (1) and (2) are methyl groups.   X in general formula (1) and (2) is an oxygen atom, The raising agent in any one of Claims 1-10.   R in general formula (1) and (2) is a propylene group, The raising agent in any one of Claims 1-11.   The raising agent in any one of Claims 1-12 whose linker group L in General formula (2) contains an azo group. The linker group L in the general formula (2) is represented by the following general formula (3)
—R ⁴ —Ar ¹ —N═N—Ar ² —R ⁵ — (3)
(Wherein R ⁴ and R ⁵ each independently represent a linear or branched alkylene group having 1 to 5 carbon atoms, Ar ¹ and Ar ² each independently represent an arylene group, and an azo group Is thin or anti-configuration.)
The elevating agent according to claim 13, which is a group represented by the formula: The raising agent according to claim 14, wherein R ⁴ and R ⁵ in the general formula (3) are methylene groups, and Ar ¹ and Ar ² are p-phenylene groups.   The raising agent according to claim 14 or 15, wherein the azo group in the general formula (3) has a syn configuration.   A melting temperature (Tm) increasing agent for a DNA molecule comprising the naphthyridine carbamate dimer derivative according to any one of claims 1 to 16, comprising a GG sequence in which two guanines (G) are continuous, and G A method of increasing the melting temperature (Tm) of a DNA molecule in a solution by adding it to a solution in which a DNA molecule having a G mismatch and at least one other mismatch is present.   A solution in which two guanine (G) has a continuous GG sequence and a DNA molecule having a GG mismatch and at least another mismatch is present at a temperature equal to or higher than the melting temperature (Tm) of the DNA molecule. The melting temperature (Tm) of the DNA molecule in the solution is increased by adding a melting temperature (Tm) increasing agent for the DNA molecule comprising the naphthyridine carbamate dimer derivative according to any one of claims 1 to 16. And then hybridizing the DNA molecule. The following general formula (4)

(In the formula, each of R ¹ and R ² independently represents a hydrogen atom, a hydrocarbon group having 1 to 5 carbon atoms, or one or more carbon atoms of the hydrocarbon group having 1 to 5 carbon atoms, Represents a group substituted by an atom selected from the group consisting of oxygen atoms, R represents a linear or branched alkylene group having 1 to 10 carbon atoms, X represents an oxygen atom or a sulfur atom, and L represents The following general formula (3)
—R ⁴ —Ar ¹ —N═N—Ar ² —R ⁵ — (3)
(Wherein R ⁴ and R ⁵ each independently represent a linear or branched alkylene group having 1 to 5 carbon atoms, Ar ¹ and Ar ² each independently represent an arylene group, and an azo group Is thin or anti-configuration.)
The group represented by these is shown. )
A naphthyridine carbamate dimer derivative represented by: The naphthyridine carbamate dimer derivative according to claim 19, wherein R ¹ and R ² in the general formula (4) are each independently an alkyl group having 1 to 5 carbon atoms. The naphthyridine carbamate dimer derivative according to claim 20, wherein R ¹ and R ² in the general formula (4) are methyl groups.   The naphthyridine carbamate dimer derivative according to any one of claims 19 to 21, wherein X in the general formula (4) is an oxygen atom.   23. The naphthyridine carbamate dimer derivative according to any one of claims 19 to 22, wherein R in the general formula (4) is a propylene group. The naphthyridine carbamate dimer derivative according to any one of claims 19 to 23, wherein R ⁴ and R ⁵ in the general formula (3) are methylene groups, and Ar ¹ and Ar ² are p-phenylene groups.   A melting temperature (Tm) increasing agent for a DNA molecule comprising the naphthyridine carbamate dimer derivative according to any one of claims 19 to 24, wherein the guanine (G) has a continuous GG sequence, and GG A method of increasing the melting temperature (Tm) of a DNA molecule in a solution by adding the DNA molecule having a mismatch and at least one other mismatch to the solution.   A solution in which two guanine (G) has a continuous GG sequence and a DNA molecule having a GG mismatch and at least another mismatch is present at a temperature equal to or higher than the melting temperature (Tm) of the DNA molecule. The melting temperature (Tm) of the DNA molecule in the solution is increased by adding the melting temperature (Tm) increasing agent for the DNA molecule comprising the naphthyridine carbamate dimer derivative according to any one of claims 19 to 24. A method of hybridizing the DNA molecule. 27. The method according to claim 25 or 26, wherein the method according to claim 25 or 26 is by syn-anti isomerization of an azo group by irradiation with light.

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