JP3157053B2 - Method for identifying the binding mode of a DNA agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for specifying a binding mode of a DNA substance.

[0002]

2. Description of the Related Art DNA is a substance that carries essential biological information for living organisms. Above all, replication and transcription of DNA are indispensable for the survival of an organism.

[0003] DNA was discovered in 1871 from the sperm of trout in the Rhine, and in 1943 it was proved that DNA was a genetic material capable of converting bacterial heritable traits. further,
In 1953, it was hypothesized that DNA forms a complementary double-helix structure.
Research on A has accelerated.

[0004] With the progress of research on these DNAs, many of the various diseases that have afflicted humans until now have become D-type.
It has been revealed that it is related to NA. Among them, as the mechanism of cancer generation becomes clear,
It has turned out to be an NA level disease. Therefore, many drugs (anticancer drugs) used for cancer treatment act on DNA. Many of these DNA-acting anticancer agents are inhibitors of DNA replication and transcription, and their effects are known to stop the growth of cancer cells or lead to necrosis. However, most of the anticancer drugs have problems such as strong side effects and a low selectivity between normal cells and cancer cells. In recent years, there have been many attempts to discover or develop new drugs with small side effects and high selectivity. As the mechanism of action, such as the structure and binding mode of DNA-acting drugs, is elucidated, the molecular design of drugs will be actively pursued based on these findings, and attempts will be made to address the side effects and selectivity issues. I have. For this reason, it can be said that obtaining knowledge on the binding mode of a DNA-acting drug is extremely useful. In addition, some of the drugs discovered and created one after another have unclear binding modes. In view of the above circumstances, a simple screening method for drugs acting on DNA has been demanded.

[0005] Screening of general anticancer drugs is shown in Table 1.
It is performed in the style as shown in

[0006]

[Table 1]

[0007] The in vivo screening of Table 1 takes a long time to elucidate the efficacy. On the other hand, in vitro screening has no direct effect on cancer cells, but it is advantageous when a large number of screenings have to be performed because the results can be known in a short time. At present, primary screening of a large number of DNA-acting drugs being developed requires a large number of screenings in a short period of time, so it seems appropriate to carry out in vitro. As mentioned above, recently, based on the knowledge on the binding mode of existing drugs, there are many attempts to enhance the drug efficacy by methods such as molecular design and chemical modification, and many new drugs are newly discovered and developed. Exists.

[0008] By the way, there are various binding modes of such a DNA-acting drug. Here, a commonly known drug will be described as an example. The binding modes are roughly classified into the following four.

[0009] Major groove, minor groove of DNA molecule
Groove) (distamycin, netropsin, etc.). Those that intercalate into DNA (actinomycin D, daunomycin, etc.). Those that bind to DNA side chains (mitomycin C, cisplatin, etc.). Those that bind to DNA and cut the DNA like enzymes (such as pleomycin).

By the way, the present inventors have proposed DNA
Research has been underway to apply a fluorescent substance intercalating as a probe to primary screening of anticancer and antiviral agents. DN using a DNA-binding fluorescent probe such as acridine orange and ethidium bromide
A detection and analysis methods of A are widely used in recent genetic engineering because of their high sensitivity and simplicity.

The fluorescent probe method using a fluorescent intercalator is now widely used as one of DNA detection methods and analysis methods. The reason is that there is an advantage that the operation is simple because no radioisotope is used, and that expensive equipment is not required. As such a fluorescent probe, ethidium bromide, acridine orange and the like are well known. These fluorescent dyes uniformly have a planar structure composed of an aromatic ring or the like, are inserted (intercalated) between base pairs of DNA, and the fluorescence characteristics are changed by intercalation. In many cases, the fluorescence intensity greatly increases. It is common to utilize this increase in fluorescence intensity to determine the presence or absence of DNA and to quantify DNA.

[0012]

The use of a fluorescent probe makes it possible to analyze a DNA substance to some extent. However, the method using this fluorescent probe makes it impossible to perform a trace analysis or limits the analytical accuracy. For this reason, there has been a demand for a more advanced method of analyzing a DNA agent.

Therefore, the present invention provides a DNA
It is an object of the present invention to provide a method for identifying a binding mode for the analysis of a DNA agent, focusing on the mode of action of the agent on DNA, that is, the diversity of binding modes.

[0014]

A solution for the] Recently, metal complexes attached to DNA ruthenium complex (such as Ru (phen) ₃ ²⁺⁾ has attracted attention. This ruthenium complex has three ligands with a planar molecular structure, one of which is known to bind to DNA by intercalating into DNA (JKBarton, Sience, 233 ,
727 (1986)). In addition, among the ruthenium complexes having phenanthroline as a ligand, it is reported that luminescence is caused by electrochemical redox, and in the presence of DNA, the electrochemical reaction is inhibited by DNA, so that the luminescence intensity is reduced. (MTCarter, AJBard, Bioconjug
ate Chem., 1 , 257 (1990)). That is, it can be said that the ruthenium complex has the two properties of acting on DNA and performing electrochemiluminescence.

Investigations have shown that the ruthenium complex also has a D value similar to that of a fluorescent probe such as ethidium bromide.
The present inventors have confirmed that the present invention can be used for analysis of an agent for NA, that is, that a ruthenium complex can be used as a probe for a drug acting on DNA, and thus the present invention has been accomplished.

The present invention is shown in the following (1) to (6). (1) In the method for specifying the binding mode of a DNA substance in a sample solution to DNA, a ruthenium complex is dispersed in the sample solution, and a current is applied to the ruthenium complex to cause electrochemical emission of light. A method for identifying the binding mode of a DNA agent, comprising identifying the binding mode of the DNA agent to DNA according to the state of (1). (2) If the amount of luminescence increases due to an increase in the amount of the DNA agent, it is determined that the DNA agent has bound to the major groove of the DNA, while if the amount of luminescence does not substantially increase, other than the major groove (1) The method for identifying a binding mode of a DNA substance according to (1) above, wherein (3) The method for identifying a binding mode of a DNA substance according to (1) or (2), wherein a ruthenium complex having a phenanthroline compound as a ligand is used as the ruthenium complex. (4) B-type DNA is used as the DNA, and the binding mode identification of the DNA agent according to any one of (1) to (3) above, utilizing the fact that a Δ-form of a ruthenium complex binds to the B-type DNA Method. (5) The method according to any one of (1) to (4), wherein the oxalate is coexistent in the sample solution. (6) The DN according to any one of (1) to (5) above, wherein a potential step method is used as a method for applying the current.
Method for specifying the binding mode of A-active substance.

[0017]

As described above, the ruthenium complex used as a probe acts on DNA and emits light, so that
Since the active substance can be analyzed and its luminescence is electrochemiluminescence, electrical control is possible. Therefore, systematization of the device is facilitated, and analysis of a DNA substance can be performed accurately and efficiently.

[0018]

DETAILED DESCRIPTION OF THE INVENTION In the present invention, a ruthenium complex which is an electrochemiluminescent probe is used as a probe for analyzing a DNA substance. Ruthenium complexes have three ligands. There are two enantiomers (Λ- and Δ-isomers) depending on the difference in the binding mode of the ligand. On the other hand, DNA has three isomers of type B, type A and type Z. B-type DNA is the most common type of DNA in vivo, and has a right-handed spiral with a wide major groove and a narrow minor groove. A
The type DNA has the same right-handed helix, but has a feature that the molecule itself is shorter and thicker than type B, and has a deep major group and a shallow and wide minor groove. However, unlike B-type and A-type, Z-type DNA has a left-handed spiral structure, and has a shallow major groove and a deep and narrow minor groove. Barton et al. First reported that the Δ-form of the two enantiomers of the ruthenium phenanthroline complex was B
Binding to type DNA (JKBa
rton, Sience, 233 , 727 (1986)). The binding mode is shown in FIG. The two figures of the ruthenium complex bound to type B DNA are molecules in the same state, each viewed from an angle rotated 90 °. The complex located at the top of the figure is a Δ-form, and one of the three ligands is bound in a state intercalated from the major groove side (right and left in the figure). At the bottom of the figure is a solid, which is electrostatically coordinated with the minor groove. Therefore, the more stable Δ-form preferentially binds to B-type DNA.
The reason that the Δ-isomer can be intercalated and the Λ-isomer cannot be thought to be because the remaining two ligands that do not intercalate cause steric hindrance along the major groove in the Λ-isomer. Replacing the ligand from phenanthroline with a larger molecule, diphenylphenanthroline, highlighting the differences between the Δ and Λ form further increases the preference of the Δ form over B-type DNA, while the Λ form is a left-handed spiral Z It has been shown to identify type DNA. This indicates that the steric hindrance to the groove of the DNA molecule by the two non-intercalating ligands described above plays an important role in molecular recognition.
If you change the central metal from ruthenium to cobalt,
It has also been reported that this metal complex becomes capable of cleaving DNA.

The ruthenium complex is DN as shown above.
It is known as a substance that performs electrochemiluminescence in addition to the interaction with A, and in particular, there is electrochemiluminescence of a complex having bipyridine as a ligand. Ru described above (phen) ₃ ²⁺ also
Ru (bpy) ₃ ²⁺ and likewise electrochemiluminescence. Bard et al. DNA
Electrochemiluminescence Ru (phen) ₃ ²⁺ in the presence indicates that slide into decreases with increasing amount of DNA (MTCart
er, AJ Bard, Bioconjugate Chem., 1 , 257 (1990)).
It is considered that the decrease in the amount of luminescence is due to the fact that the ruthenium complex binds to the DNA and cannot react with the electrode. However, they used this ruthenium complex as a probe for DNA quantification and, as in the present invention,
Not used for analysis of NA agonists.

The above results indicate that the ruthenium complex is DN
This indicates that the probe has an interaction with A and is a substance that performs electrochemiluminescence.

The following summarizes what has been described about the properties of the ruthenium complex.

Binds to a DNA molecule. Electrochemiluminescence. Electrochemiluminescence decreases in the presence of DNA.
On the other hand, many drugs used as anticancer drugs and antiviral drugs have an interaction with DNA, such as ruthenium complexes. In other words, it can be said that the contact point between the ruthenium complex and the DNA-acting drug is that both have "interaction with DNA". Therefore, the inventors have proposed DNA
It was speculated that the drug bound to may inhibit the intercalation of the ruthenium complex. It is believed that the electrochemiluminescence properties of the ruthenium complex in a state where the intercalation is inhibited by the drug bound to DNA change depending on the binding mode of the drug. As a result, a change in the luminescence characteristics was obtained, and a knowledge of the binding mode of the drug was obtained.
We thought that it could be applied to screening for NA-binding drugs.

FIG. 2 shows the principle of the evaluation. After a predetermined amount of the drug (A, B) were bound to DNA, Ru (phen) ₃ added to the type of drug ^2+, complex binds according to the amount. That is, the binding of the ruthenium complex to the DNA bound with the drug is inhibited, and the amount of the unbound complex increases. Drug A,
Since B has a different action on DNA, the amount of unbound complex also differs. If DNA inhibits the electrochemical reaction of the ruthenium complex, the amount of unbound complex should reflect the amount of luminescence. From the above, it is considered that the luminescence characteristics change depending on the type of the drug, whereby the drug can be evaluated.

In the present invention, as described above, a ruthenium complex is used as an electrochemiluminescent probe.

[0025] The details of Ru (phen) ₃ ²⁺ electrochemiluminescence reaction mechanism to be used as a probe no necessarily clear,
Ru with the same ruthenium complex and bipyridine as ligand
(bpy) ₃ ²⁺ and is the element emits light in a similar manner. Ru (bpy) ₃
²⁺ emits light through a reaction route as shown in Chemical ^formula 1.

[0026]

Embedded image

Bard et al. Disclose Ru (phen) _{3 in the} presence of oxalate.
^It has been described that the electrochemiluminescence of ²⁺ goes through a route as shown in Chemical ^formula 2.

[0028]

Embedded image

In the reaction route shown in Chemical formula 2, the reaction of 1) triggers light emission. 1) R shown in equation
oxidation of u (phen) ₃ ²⁺ occurs at least about + 1.5V relative to the silver / silver reference chloride electrode. The electrochemiluminescence of the ruthenium complex decreases when DNA coexists, and the luminescence is
There is a relationship of decreasing according to the NA amount. This is because the ruthenium complex has bound to the DNA, 1) or 4) CO ₂ ^.− / Ru (phen) ₃ between the electrode / Ru (phen) ₃ ^{2+ due} to the bound DNA molecule in the reaction shown in equation (4) ^{. This} is probably because electron transfer between ²⁺ is suppressed. Bard et al. Ru (phe
n) Although the electrochemiluminescence ₃ ²⁺ were attempts to use the quantitation of DNA, that said the light emission amount is not large enough for the high sensitivity measurement. The present inventors evaluated this luminescence reaction not by quantification of DNA, but by evaluating the mechanism of action of a drug acting on DNA, and considering the possibility of identifying the drug in reverse. The purpose was to know.

[0030] As an example, here, Ru (phen) electrochemiluminescence characteristics of ₃ ^2+, and Ru in λDNA presence (ph
en) illustrating the emission characteristics of the ₃ ^2+.

In recent years, as knowledge about the binding mode between a DNA-acting drug and DNA has become available, attempts have been made to molecularly design and chemically modify new drugs based on these findings. It is being actively performed.

As an example of developing a drug having a new function by designing a molecule based on a binding mode, there is an improvement of bleomycin. In addition, examples of attempts to solve problems such as side effects by performing chemical modification based on the binding mode include cisplatin, a complex of platinum, which is known as a drug with a wide range of anticancer effects. Used to treat cancer. FIG. 3 shows the chemical structures of these anticancer agents and antiviral agents.

Such a chemical approach to drug development has received a great deal of attention in recent years. The present invention relates to DNA
The present invention relates to an analysis using an electrochemiluminescent probe as a means for obtaining information on the binding mode of an active drug.

Measurement of Electrochemiluminescence in the Presence of a DNA Agonist As described above, when a ruthenium complex binds to DNA,
Light emission is reduced due to steric hindrance of the DNA molecule. When a drug that binds to DNA coexists in such a system, the ruthenium complex and the drug both bind to DNA, and therefore, it is considered that they interfere with each other to bind to DNA. On the other hand, the amount of electrochemiluminescence of the ruthenium complex in the presence of DNA is presumed to represent the amount of the ruthenium complex in the free state. As will be described in detail later, for example,
FIG. 4 shows the electrochemiluminescence characteristics of the ruthenium complex in the presence of λDNA.

When a ruthenium complex is previously added to DNA to which a drug is bound, new binding of the complex is inhibited by the drug that has already bound to the DNA.
At this time, the amount of the ruthenium complex in the free state seems to have increased as compared with the case where only the complex bound to DNA. Since the amount of complex in the free state can be quantified by electrochemiluminescence, an increase in luminescence will correspond to an increase in the amount of free ruthenium complex whose binding to DNA has been inhibited by the drug. The inhibition of the binding of the complex to DNA by such a drug should differ depending on the binding mode of the drug.

FIG. 5 shows a measurement principle based on this hypothesis. The case A) in the figure is the electrochemiluminescence when only the ruthenium complex is present in the system. The bar grab on the right side of the figure indicates the light emission amount in the case of A). Here, based on this light emission amount, the light emission amount when DNA and drug coexist is to be relatively evaluated. In the case B) of the figure, luminescence is observed in the presence of DNA. As shown in the above figure, when the ruthenium complex binds to DNA, steric hindrance occurs, so that the amount of luminescence decreases as shown in the right graph of FIG. 5B). The luminescence amount at that time indicates the amount of the ruthenium complex in a free state. C) and D) show luminescence in the presence of DNA on which drugs A and B have previously acted. In each of the systems C) and D), drugs A and B having different binding modes are assumed. When DNA is mixed with a drug, drugs A and B bind to the DNA according to the respective binding modes. If the ruthenium complex is subsequently added,
The binding of the ruthenium complex is inhibited by the drugs A and B already bound to the DNA, and the number of complexes in the free state is B)
(E.g., one free complex in B), two in C) and four in D). Therefore, the light emission amount also increases as compared with B) (the state is schematically shown by a bar graph at the right of each figure). C) light emission amount,
There is a difference in the light emission amount of D). This is presumably because drugs A and B have different binding modes, and drug A has a weaker effect of inhibiting binding of a ruthenium complex to DNA than drug B, so that a difference appears in the number of free complexes. That is, the difference in the amount of light emission indicates the difference in the binding mode (in this case, the difference in the number of bonds), and by using this, the binding mode of the drug can be specified.

Experimental Example Hereinafter, an experimental example for confirming the operation of the present invention will be described with reference to the accompanying drawings.

Measurement Method and Apparatus The following reagents and measurement apparatus reagents were used. λ DNA (from bacteriophage λ cI857S7) (Takara Shuzo Co., Ltd. poly (dA)
· Poly (dT) and poly (dG) · poly (dC ) Boehringer Mannheim Yamanouchi ^{_{(Ltd.)) Ru (phen) 3 2+}} (Tris (1,10-phenan
throline) -ruthenium (II) chloride hexahydrate) (Al
drich Chem. Co.) was prepared.

FIG. 6 shows an outline of the measuring apparatus. As shown in the figure, the measuring device includes a dark box 1, a photomultiplier tube (photomultiplier tube) 2, a power supply 3, a luminescence measuring unit 4 composed of a photon counter, a potentiostat 5,
A function generator (function generator) 6, an electrochemical system such as an electrochemical cell, a luminescence measuring unit, and a potential change output from the electrochemical system;
And an oscilloscope 7 for observing the light emission amount, and a recording unit 8 composed of an XY recorder. The electrochemical cell 9 in the dark box 1 controlled a three-electrode system consisting of a platinum working electrode, a platinum counter electrode, and an Ag / AgCl reference electrode. The electrochemical cell 9 was arranged immediately before the measurement window 2a of the photomultiplier tube 2 as shown in the figure.

Photomultiplier tube (R464),
The photon counter (C767), power supply (752-01), and other light emission measurement units are manufactured by Hamamatsu Photonics KK, and the potentiostat (2090-LN) is manufactured by Hokuto Denko KK, function generator (HB-105). Is manufactured by Toho Giken Co., Ltd., oscilloscope (VC-6020) is manufactured by Hitachi, Ltd., XY recorder
(WX2400) manufactured by GRAPHTEC was used.

The quantification of the ruthenium complex and DNA was carried out using an ultraviolet-visible spectrophotometer (Ubest-30, JASCO Corporation).

Luminescent Properties of Electrochemiluminescent Probe Ru (phen) ₃ ²⁺ : tri (1,
10-phenanthroline) -ruthenium (II) chloride hexahydrate) (Tris (1,10-phenanthroli)
ne) -ruthenium (II) chloride hexahydrate) was used. In the present invention, it is desirable to use a potential step method rather than a potential scanning method as an electrochemical excitation method for measuring light emission. FIG. 7 shows a schematic diagram of a light emission pattern according to the potential step method. At this time, the accumulated light emission amount for 10 seconds at a potential of 1.5 V (shaded area in the figure) is 10
(CPS: Count Per Seco)
nd) was defined as the luminescence value.

All reagents such as ruthenium complex are
s-Buffer (50 mM Tris-HC1, 50 mM NaCl, 15 mM Sodium Oxalat
e pH 7.3) and measured the luminescence. After each measurement, the electrode was washed with distilled water and then washed by a potential application method. Then, it glowed red with a gas burner.

Emission Characteristics of Electrochemiluminescence Probe in the Presence of DNA The concentration of the ruthenium complex for examining the luminescence behavior in the presence of DNA is, for example, 1 μM. The electrochemical excitation is performed in a potential step, and the emission is performed using a ruthenium complex (1 μm).
The evaluation was based on the relative value to the luminescence when only M) was present in the measurement system. After mixing the ruthenium complex and DNA, at 37 ° C
After 12 hours or more, the sample solution was used for luminescence measurement.

Actually, the following three kinds of DNA samples were selected. Λ DNA as a standard DNA model, and poly (dA) / poly (dT) to clarify base sequence specificity in binding between ruthenium complex and DNA, and
poly (dG) and poly (dC) were used. DNA quantification was calculated based on the absorbance (260 nm).

Results and Discussion Luminescence Characteristics of Electrochemiluminescence Probe Electrochemiluminescence was performed by the potential scanning method (0V → 2V → 0V). FIGS. 8 and 9 show the results obtained by the potential scanning method. In addition, referring to the emission potential obtained by the potential scanning method, electrochemical excitation (excitation is 1.5 V) by the potential step method (0 V → 1.5 V).
For 10 seconds). FIG. 10 shows the step potential and the emission pattern in the absence of oxalate, and FIG. 11 shows the oxalate (1
Fig. 1 shows the change in potential and the emission pattern in the presence of 5mM).
2 contains oxalate and DNA (15.3μ in base pair conversion)
M) shows the results under coexistence. The luminescence increased significantly in the presence of oxalate. In the presence of oxalate, a radical (CO ₂ ^.- ) is generated from the oxalate, which reacts with a trivalent ruthenium complex to produce Ru (phen), an exciter that emits light.
₃ ^{2 + *} is generated. On the other hand, when oxalate does not exist, the trivalent ruthenium complex transfers electrons to and from the solvent to generate a divalent exciter. 2 necessary for emitting light
Since the exciton of the valence complex is efficiently generated by radicals in the presence of oxalate, the amount of light emission also increases. Also D
Although the luminescence pattern was not changed in the presence of NA (FIG. 12), the amount of luminescence was significantly reduced as compared with the absence of DNA (FIG. 11). In the measurement of the amount of light emission, when the average amount of light emission per second at +1.5 V (unit: cps) was measured, a stable amount of light emission was obtained.

Dependence of Light Emission on Oxalate Concentration Oxalate in the sample solution was varied as shown in Table 2 to determine the dependence of light emission on oxalate concentration.
Table 2 shows the results.

[0048]

[Table 2]

As shown in Table 2, the luminescence amount increased dramatically as the oxalate concentration increased. However, at concentrations higher than 15 mM, no significant increase in luminescence was observed. Therefore, the oxalate concentration in the luminescence measurement was 15 mM.
Decided to set.

The dependence of the luminescence on the concentration of the ruthenium complex was also clarified. The results are shown in FIG. 13 (without oxalate) and FIG. 14 (with oxalate (15 mM)). By comparing FIG. 13 and FIG. 14, it was found that the luminescence amount in the presence of oxalate reached 100 times or more that in the absence of oxalate. In addition, in the presence of oxalate, it was found that luminescence was about 1000 (cps) at 1 μM of the ruthenium complex. Therefore, when used for analysis on the binding mode of DNA and drugs, the buffer solution contained oxalate (15 mM). Tris buffer solution (pH 7.3)
M, potential step method was used for electrochemical excitation.

Luminescent Properties of Ru (phen) ₃ ^{2+ in} the Presence of DNA In the present invention, the binding mode of a drug is estimated based on the change in the luminescent properties of a ruthenium complex in the presence of a DNA to which a drug has been previously bound. I do. That is, the ruthenium complex is used as a probe for a drug acting on DNA.
Therefore, here, the relationship between the interaction between the ruthenium complex / DNA and the electrochemiluminescence characteristics was analyzed.

As a DNA sample, λ DNA having a clear base sequence and readily available was used. FIG.
The electrochemiluminescence characteristics of the ruthenium complex in the presence of DNA were shown. In this figure, the horizontal axis represents the molar concentration (μM) of DNA converted per base pair, and the vertical axis represents the amount of luminescence when only 1 μM of the ruthenium complex is present (about 1000 cps).
Is the relative value of the light emission when 100 is assumed.

The electrochemiluminescence of the ruthenium complex decreases until the amount of DNA base pairs reaches about 20 μM. Above this, no decrease in luminescence was observed. The reason for this is electron transfer suppression of the electrode by steric hindrance the ruthenium complex is bound to DNA, 2 divalent excitation body ^{_{([Ru (bpy) 3 2+}} ]
It is considered that the generation amount of ()) decreases, and therefore the light emission amount also decreases. Also, from the figure, the DNA base pair amount was 20 μM.
From the above, it can be seen that the decrease in the amount of light emission reaches saturation. Since the concentration of the ruthenium complex is 1 μM, the saturation binding ratio between the DNA base pair and the complex is considered to be about 20: 1.

As described above, for a B-type DNA such as λ DNA, of the two isomers of the ruthenium complex (Λ, Δ-isomer), it is assumed that the Δ-type complex mainly binds to the B-type DNA. ing. Here, the ruthenium complex used is a racemic body. In addition, it can be seen from the results of FIG. 4 that the luminescence amount is reduced by about 50% when the bond of the complex reaches saturation. From this, half of the total number of complexes, that is, Δ
It is inferred that only the body binds to DNA and the complex in the free state is a Λ body. Considering these matters, it is considered that the saturation bond ratio of only the Δ-form is 40: 1, that is, one Δ-type complex binds to 40 base pairs. In the case of the B-type double helix DNA, the number of base pairs required for one helix is 10 base pairs, so it can be said that the ruthenium complex is bonded at a ratio of one to four helices. Here in FIG. 1 Ru (phen) ₃ ²⁺
Referring to the computer graphic of the state in which the DNA and DNA are bound, one molecule of the bound ruthenium complex covers almost half of the major group of one helix (10 bp). In other words, "to bind one to four helices (40 bp)" can be paraphrased as "to occupy five base pairs out of 40 base pairs (corresponding to a major group of helices)".

Relationship between DNA Type and Luminescent Properties It has already been described that ruthenium complexes intercalate and bind to DNA. However, the nucleotide sequence specificity of the binding of the ruthenium complex to DNA is not always well understood. To apply the ruthenium complex to the evaluation of the binding mode of drugs, it is important to obtain knowledge about the nucleotide sequence specificity. Therefore, poly (dA), poly (dT), and poly
(dG) · poly (dC) was selected, and the amount of luminescence in the coexistence thereof was measured. Table 3 shows the results.

[0056]

[Table 3]

As can be seen from this table, the amount of luminescence decreases in the presence of DNA, irrespective of the type, relative to the amount of luminescence of the control. However, the amount of reduction is DN
A difference was observed depending on the type of A. poly (dA) ・ poly (d
In the presence of (T), the relative luminescence was 59.0, whereas poly (d
G) ・ poly (dC) is as large as 73.4. On the other hand, λ DNA having any bases of adenine, thymine, guanine and cytosine
The light emission amount in the coexistence is 66.7, which can be said to be an intermediate value of the relative light emission intensity of both. The higher the light emission amount, the smaller the amount of the bonded ruthenium complex. These results show that the ruthenium complex strongly binds to DNA consisting of AT. However, it can be said that the specificity of the base sequence of the binding of the ruthenium complex is not so large as can be understood from the fact that it binds to a considerable amount of DNA composed of GC.

It is considered that the relationship between the DNA base sequence and the luminescence reflects local structural variation due to the base sequence of the B-type helical DNA molecule. In the case of B-type DNA, helical twist between two adjacent base pairs, prohera twist of each base pair (the two bases in a pair rotate in opposite directions around the long axis), etc. There are local structural fluctuations. The ruthenium complex intercalates one of the three ligands with DNA and the other two D
It is said that they bind along the direction of the large groove (major groove) of the NA molecule (see FIG. 1). If poly
If a difference in the structure of the major groove appears between AT and poly GC due to subtle variations in the local structure, the remaining two non-intercalated ligands are joined to the major group of poly AT and poly GC. There should be a difference in gender. Since the difference in bonding property is considered to be directly reflected on the number of bonds of the complex, it is considered that a difference also appears in light emission.

The above is summarized as follows. Ru (phen) ₃ ²⁺ is electrochemiluminescence, the emission is significantly increased in the presence of oxalate. Stable and sufficient light emission could be obtained by performing electrochemical excitation by the potential step method. The emission of the ruthenium complex in the presence of λDNA is D
It decreased with increasing NA amount. The decrease in luminescence in the presence of DNA is 20 times higher than that of complex
Saturation was reached when the A amount was added. Taking this result and the binding mode of the ruthenium complex into consideration, it is considered that the ruthenium complex occupies a major groove corresponding to 5 base pairs out of 40 base pairs. It was found that the decrease in the amount of luminescence in the presence of DNA was different depending on the base sequence. This result seems to indicate that the binding of the ruthenium complex to DNA is slightly different depending on the base sequence.

[0060] Since the above as, Ru (phen) ₃ ²⁺ is D
It was shown that its binding to NA reduced its electrochemiluminescence in the presence of DNA. Therefore, DNA in the measurement system
When the drug to bind coexist, the drug and Ru (phen) ₃ ²⁺ are both bind to DNA, the effect was found to be reflected as light-emitting characteristics change in some way. Therefore, leading the Ru (phen) ₃ ²⁺ to the present invention used as a probe to drugs that bind to DNA.

Electrochemistry in the Presence of a DNA Agent
Solutions for luminescence properties of luminescent probes and drug binding modes
The analysis reagent and measuring instruments reagents used were as follows. Daunomycin (D
aunomycin), actinomycin D, cisplatin (cis-DDP: cis-dichloro diammine platinum)
(II)) is what the SIGMA CHEMICAL Co., chromomycin A ₃ (ChromomycinA ₃₎ was used as manufactured by Wako Pure Chemical Industries, Ltd.. The chemical structures of these four drugs are shown in FIG. The histone protein (Histone from calf thymus) used was manufactured by Boehringer Mannheim Yamanouchi. [lambda] DNA, and Ru (phen) ₃ ²⁺ is the same as that used in the previous.

The same measuring device as that shown in FIG. 6 was used. The ultrafiltration membrane used to separate the drug bound to the DNA from the unbound drug used was a low-adsorption molcut II (UPF1 LGC fractionated molecular weight of 10,000) manufactured by Nippon Millipore Industries, Ltd. Ruthenium complex, DN
A and a UV-visible spectrophotometer (U best-30) manufactured by JASCO Corporation were used for quantification of A and drug (excluding cisplatin). Platinum (Pt) contained in cisplatin was quantified using an atomic absorption spectrophotometer (AA-660) manufactured by Shimadzu Corporation and a hollow cathode lamp for Pt manufactured by Hamamatsu Photonics KK as a light source.

Method for Measuring Electrochemiluminescence in the Presence of a DNA Agonist The chemical procedure is shown in Chemical Formula 4.

[0064]

Embedded image

The amount of luminescence measured in Chemical formula 4 is the luminescence of the ruthenium complex in the presence of the drug bound to DNA.
Is the luminescence when only the drug is present and is a control for. Here, in the presence of a ruthenium complex (1 μM), drugs were added at various ratios to DNA, and the luminescence was measured. Luminescence is ruthenium complex
(1 μM) was evaluated relative to the amount of luminescence when present only. The evaluation was performed using a graph in which the relative luminescence was plotted against the amount of the drug. As the DNA agonists, it was used cisplatin, daunomycin, actinomycin D, and chromomycin A _3.

Measurement of the binding ratio between each drug and DNA Based on the principle shown in FIG. 17, the binding of the ruthenium complex is inhibited by increasing the amount of drug added until the binding between DNA and drug reaches saturation. It is expected that free ruthenium complexes will continue to increase. However, after drug binding has reached saturation, the inhibitory effect of the ruthenium complex on DNA binding appears to be different.
For this reason, it is necessary to determine the molar ratio (saturation bond ratio) between the DNA and the drug when the binding between the DNA and the drug reaches saturation.

FIG. 15 shows an outline of the measuring method. As shown in the figure, the measurement is performed for both the case where DNA is present and the case where DNA as a control is not present.
First, a drug is added to a fixed amount of DNA so as to have a predetermined concentration ratio, and the mixture is left at 37 ° C. overnight. At this time, only the same amount of drug is added as a control. Thereafter, the drug bound to DNA and the free drug are separated by mole cut, and the free drug contained in the filtrate is quantified. On the other hand, in the absence of DNA, all the drug can pass through the membrane, so the amount of drug in the filtrate is the same as the amount of drug added initially. The difference between the amounts of drug contained in both filtrates indicates the amount of drug bound to DNA.

When the amount of drug to DNA is gradually increased, the drug binds to DNA and remains on the membrane until the binding of drug to DNA reaches saturation. Therefore, no drug is detected in the filtrate. However, after the binding reaches saturation, some of the added drug cannot be bound to DNA and flows out. Therefore, the amount of free drug in the filtrate is DN
It is believed to be the same as the amount of drug added beyond the saturated binding of the drug to A. That is, the amount of drug in the free state detected in the filtrate seems to increase at the saturation binding ratio of drug to DNA. The saturation bond ratio was determined by measuring the increase.

[0069] Note that quantitative actinomycin D drugs, daunomycin, with absorbance method for chromomycin A _3, for cisplatin were performed by atomic absorption spectrometry.

Cisplatin, daunomycin, actino
Luminescence in the presence of mycin D and chromomycin A ₃
Consideration on properties and binding mode of cisplatin cisplatin (cis-dichlorodiammine platinum (II): ci
FIG. 16 shows the results of luminescence measurement performed when a ruthenium complex was allowed to coexist with DNA on which s-DDP) had previously acted. The figure also shows the structural formula of cisplatin in addition to the drug amount-luminescence curve. Cisplatin on the right side of the figure is a complex of platinum and is a compound having a planar structure having chlorine and ammine as ligands. The horizontal axis of the graph (drug amount-luminescence curve) on the left side of the figure represents the logarithm of the concentration (μM) of cisplatin added to the system, and the vertical axis represents the luminescence amount at that time by a ruthenium complex (1 μM). It is expressed as a relative value with respect to the light emission amount when only one exists. In addition, the two curves, the upper graph is a drug amount-luminescence curve when only the drug coexists in the system,
The lower right-upper graph is a drug amount-emission curve when a DNA to which a drug is bound is present in advance.
The concentration of λ DNA is constant throughout the measurement, and is 12.0 μM in terms of base pairs.

From the above graph, when only the drug coexists, no change was observed in the amount of luminescence even when the concentration of the drug was increased, as can be seen from the amount of drug-luminescence curve (upper graph (DNA (-))). That is, cisplatin itself did not affect the electrochemiluminescence of the ruthenium complex.However, in the presence of DNA in which a drug had been acted on in advance, the drug amount-luminescence curve (lower graph (DNA (+)) Unbound D
In the co-presence of NA alone, the luminescence decreased similarly to the above results, but after that the luminescence increased with increasing drug concentration. The increase in luminescence continued until the relative luminescence of 100 (the luminescence when only the ruthenium complex (1 μM) was used). That is, when only DNA (12.0 μM in base pair conversion) coexists without adding cisplatin, the relative luminescence was 65.3%.
On the other hand, the relative luminescence increased as the amount of cisplatin acting on DNA was increased, and the relative luminescence increased to 91.4 when cisplatin was 22.0 μM. As a result, cisplatin bound to DNA is converted to DN.
It was suggested that the binding of ruthenium complex by A was suppressed, and as a result, light emission increased. That is, as the amount of cisplatin added increases, the amount of ruthenium complex that is sterically hindered by the DNA molecule decreases, so that light emission also increases. When the ruthenium complex cannot bind to DNA due to the cisplatin bound to DNA, the relative luminescence is considered to be 100 (the luminescence when both DNA and cisplatin are not present).

As described above, if cisplatin inhibits the binding of the ruthenium complex to DNA, when the binding of the cisplatin to DNA reaches saturation, the binding of the ruthenium complex is not inhibited any more after saturation, and the amount of luminescence does not increase. The increase is likely to stop there. As the saturated bond ratio, the maximum bond amount in the neighborhood exclusion rule model was obtained as the saturated bond ratio. The neighbor exclusion rule is that when a substance that intercalates with DNA binds, new intercalation of a base pair adjacent to the base pair to which the substance binds is inhibited. The maximum binding amount based on the neighborhood exclusion rule is a case in which one binding occurs every two base pairs, and in this measurement, it corresponds to a cisplatin concentration of 6 μM which is half of the DNA amount. In the graph on the left of the figure, the maximum binding amount based on the neighborhood exclusion rule is the drug concentration indicated by the dotted line. According to this, an increase in light emission is observed even after the dotted line indicating the maximum binding amount. This indicates that cisplatin in excess of the limit binding amount binds and inhibits the binding of the ruthenium complex. That is, cisplatin is used as a DNA at a frequency of one or more per two base pairs exceeding the critical binding amount in the neighborhood exclusion rule model.
It is considered to be connected to.

By the way, as shown in FIG. 1, the ruthenium complex binds to a major groove of DNA, and one of its ligands is said to intercalate with DNA. In order for cisplatin to inhibit the binding of this ruthenium complex to DNA, it must be bound or intercalated at least in the major groove, like the ruthenium complex. Cisplatin binds to guanine (G) and adenine (A) residues of DNA, and its binding mode has conventionally been determined by an intramolecular reaction (DN
Although two modes have been proposed, a form of binding to one strand of A) and an intermolecular reaction (a form of linking two strands of DNA), the details have not been clarified yet (see FIG. 17). ).

Here, when an attempt was made to electrochemically reduce the cisplatin bound to the DNA, it was found that in the state bound to the DNA, the reduction reaction of the cisplatin was remarkably suppressed by steric hindrance of the DNA molecule. ing. This result suggests that the binding mode of cisplatin corresponds to an intermolecular reaction (Interstrand Binding) shown in FIG. On the other hand, the results of this example show that the binding mode of cisplatin is binding in a major groove or intercalation. Considering this, it is considered that the binding mode of cisplatin corresponds to the intermolecular reaction in FIG. This is consistent with the results of the electrochemical reduction described above. From the above,
Cisplatin binds or intercalates with major grooves in DNA, and that binding has been shown to occur more than once every two base pairs without obeying the neighborhood exclusion rule.

Daunomycin Daunomyci
FIG. 18 shows the results of luminescence measurement performed when a ruthenium complex was allowed to coexist with DNA to which n) had been acted in advance.
The horizontal axis and vertical axis of the graph (drug amount-luminescence curve) on the left side of the figure are the same as those in the case of cisplatin (FIG. 16). The upper graph and the lower graph also show a drug amount-luminescence curve in the absence and presence of DNA, respectively, as in FIG. Daunomycin is an antibiotic consisting of an anthracycline ring and daunosamine as shown in FIG. 18. The B and C rings of anthracycline intercalate into DNA, and the amino group of daunosamine is electrostatically bound to the phosphate group of DNA. Thus, it is known that they strongly bind to the AT (adenine thymine) sequence of DNA and inhibit the reaction of DNA polymerase and RNA polymerase. Also, DN
The amount of A is 13.0 μM in terms of base pairs.

From the graph (DNA (-)), it was found that when only daunomycin coexists, the amount of luminescence remarkably decreases as the amount of drug increases unlike in the case of cisplatin. It is considered that the reason for the decrease in the light emission amount is that the light emission of the ruthenium complex is absorbed by daunomycin or that light energy is transferred between daunomycin and the ruthenium complex. On the other hand, under the coexistence of DNA to which daunomycin had been acted before, different tendencies were observed between the first half and the second half of the graph. In the first half of the graph, as in the case of cisplatin, an increase in luminescence was observed with an increase in the amount of drug, and in the second half of the graph, conversely, a decrease in luminescence was observed with an increase in the amount of drug.

This phenomenon is considered as follows. That is, the first half of the graph is a process before the binding of daunomycin to DNA reaches saturation. In this process, the binding of the ruthenium complex to DNA by daunomycin occurs. At this time, an increase in luminescence is observed as in the case of cisplatin. In the second half of the graph, daunomycin and D
This is a process after the binding to NA reaches saturation. Daunomycin added in excess of the saturation amount cannot bind to DNA and becomes free, reducing the light emission of the ruthenium complex. Therefore, the saturation binding ratio was determined, and it was determined whether or not the saturation binding ratio coincided with the transition between the first half and the second half of the drug-luminescence curve.

FIG. 19 shows the result of the determination of the saturation bond ratio.
The horizontal axis of this graph represents the amount of daunomycin bound to DNA as a relative value when the amount of DNA in base pair is set to 1. The vertical axis represents the concentration of daunomycin contained in the filtrate when daunomycin and DNA were mixed at a predetermined ratio and then subjected to ultrafiltration. Solid line graph
(DNA (+)) is the result of mixing DNA and drug and then filtering. As a control, when only the drug equivalent to the case of mixing with DNA was added, and the mixture was filtered, the amount of the drug in the filtrate at that time increased linearly as the amount of daunomycin added increased. This indicates that the drug hardly adsorbs to the ultrafiltration membrane.

On the other hand, when DNA is present, FIG.
As shown in Fig. 5, even when the relative amount of daunomycin to DNA increases, almost no drug flows out until the amount exceeds a certain amount, and after that amount, the drug flows out linearly. This means that all the mixed drugs bind to DNA until the saturation binding ratio is reached, so that there is no free drug, and if the drug is more than that, it cannot be bound and flows out into the filtrate. It is considered something. Therefore, it is considered that the outflow start point corresponds to the saturation bond ratio. The relative amount of daunomycin to DNA at the onset of efflux was 0.38. That is, it was found that one daunomycin binds to 2.63 base pairs.

When the plot was re-plotted in FIG. 20 using this saturation bond ratio, a good agreement was found with the transition between the previous and latter half. Note that the saturation bond ratio is represented by a chain line in the graph. Before and after this dashed line, it is considered that there is a remarkable change between the amount of bound daunomycin and the amount of free daunomycin as shown in FIG. That is, daunomycin binds according to the amount of drug added before the saturation binding ratio, but becomes constant after the saturation binding ratio. On the other hand, free daunomycin is hardly present in the system before the saturation binding ratio because most of the added drug binds, but when the ratio exceeds the saturation binding ratio, it increases according to the amount of the added drug.

In consideration of the above, if the effect of reducing light emission due to free daunomycin is removed, correction can be made after the saturation binding ratio in the graph shown in FIG. The corrected graph is shown in FIG. According to the graph after the correction, the light emission increases up to the saturation binding ratio and does not change thereafter. In addition, the increase in light emission reaches a relative light emission amount of about 90 or more. This indicates that daunomycin efficiently suppressed the binding of the ruthenium complex to DNA. At this time, if the binding position of the ruthenium complex and the binding position of daunomycin do not overlap, it is considered that the binding is not inhibited. The binding of the ruthenium complex to DNA occupies 5 out of 40 base pairs as described above. At this time, there is an empty space for about 35 base pairs. When the concentration of daunomycin is low, the ruthenium complex can bind to DNA without being inhibited (repressed) by daunomycin if it binds to such a space. However, as shown in the graph, the luminescence began to increase from a low daunomycin concentration, and the relative luminescence almost approached 100 near the saturation binding ratio. This indicates that daunomycin inhibits the binding of the ruthenium complex even at a low concentration. As described above, it has been revealed that the ruthenium complex binds more strongly to the AT sequence than to the GC sequence of DNA. As described above, daunomycin also strongly binds to the AT sequence. This suggests that daunomycin and the ruthenium complex have the same binding sites, and thus the above-described binding inhibition occurs with high efficiency.

Further, it is considered that the presence of daunosamine constituting daunomycin is important. As can be seen from the fact that the amino group of daunosamine is electrostatically bonded to the phosphate group of DNA, it is thought that the amino group is present in the groove without intercalation between base pairs. If daunosamine is present in the minor groove, the major groove is vacant and the ruthenium complex can be bound, but the high-efficiency binding inhibition as described above does not occur. Therefore, it is considered that daunosamine is present in the major groove and strongly inhibits the binding of the ruthenium complex.

To summarize the above, daunomycin inhibits (suppresses) the binding of ruthenium complex to DNA with high efficiency.
It is concluded that the binding to the sequence and the presence of daunosamine in the major groove further enhances the binding inhibitory effect.

Actinomycin D FIG. 22 shows the result of luminescence measurement when a ruthenium complex was allowed to coexist with DNA on which actinomycin D had been acted on beforehand. The graph in the figure is a drug amount-luminescence curve, in the same manner as in the case of cisplatin and daunomycin. Actinomycin D is an antibiotic consisting of a phenoxazone skeleton and two peptide lactone rings as shown in the figure. In actinomycin D, phenoxazone skeleton is intercalated into DNA, and two peptide lactone rings are oriented in opposite directions (upstream and downstream of DNA strand), respectively, and bind to minor groove to selectively perform RNA synthesis. It is said to inhibit. It is said that 5 to 6 base pairs are required for binding to DNA by intercalation and orientation to minor grooves. The binding base sequence is different from daunomycin and is considered to be a GC (guanine / cytosine) sequence. The amount of DNA in this example is 14.4 μM in terms of base pairs.

From the luminescence curve (DNA (-)) shown in the figure, when only actinomycin D was present, no significant change was observed in the luminescence even if the amount of drug was increased. This indicates that actinomycin D does not significantly affect the electrochemiluminescence of the ruthenium complex. On the other hand, the graph in the left figure (DNA (+))
More specifically, DN in which actinomycin D was previously acted on
In the coexistence of A, no increase in the amount of luminescence associated with an increase in the amount of drug acting on DNA, such as cisplatin or daunomycin, was observed. However, when the amount of actinomycin D was excessive, the amount of luminescence increased.

Next, the saturation binding ratio between actinomycin D and DNA was determined. The result is shown in FIG. The format of the graph is the same as that for daunomycin in FIG. The relative amount of actinomycin D to DNA at the saturation binding ratio was 0.22. That is, it was found that actinomycin D binds one molecule to 4.55 base pairs of DNA. Taking into account the number of salt pairs occupied by actinomycin D when binding to DNA (5 to 6 base pairs), actinomycin D is bound to all base pairs of DNA in a saturated binding state. This saturation binding ratio was applied to the drug amount-luminescence curve of FIG. 22 and represented by a dashed line.

In consideration of the above, the increase in luminescence was not observed until the saturation binding ratio was reached in the drug amount-luminescence curve, but it was shown that the luminescence increased after reaching the saturation binding ratio. Was done. Before reaching the saturation bond ratio, the DNA of the ruthenium complex is considered from the fact that no increase in luminescence is observed.
It is thought that the binding to is not inhibited (suppressed). Further, after reaching the saturated bond ratio, an increase in light emission is observed, so that it is considered that the binding of the ruthenium complex to DNA is inhibited (suppressed). If there is a difference in the inhibition of the binding of the ruthenium complex to DNA by actinomycin D before and after the saturation binding, two problems arise. That is, before reaching the saturation binding ratio, the ruthenium complex must be able to further bind even though actinomycin D has already bound to DNA. After reaching the saturation bond ratio,
Actinomycin D in a free state added in excess of the saturation amount must be able to inhibit the binding of the ruthenium complex to DNA.

First, the case before the saturation coupling will be described. The following three cases can be considered for further binding of the ruthenium complex with actinomycin D already bound to DNA. : Actinomycin D is detached from DNA by binding of a ruthenium complex. :
The ruthenium complex binds to the base pair in the gap where actinomycin D is not bound. : Actinomycin D is D
Since the ruthenium complex is bonded to the minor groove side of NA, the ruthenium complex is further bonded to the vacant major groove side. Consider the first case. When the ruthenium complex was added to the DNA to which actinomycin D was saturated and bound, and then the detached actinomycin D was separated by mole cut, the detached actinomycin D was not detected in the filtrate. From this, it seems that elimination (substitution) of actinomycin D by the ruthenium complex does not occur. In the following case, when the actinomycin D concentration is low, it is conceivable that the actinomycin D binds to the base pairs in the gap, but in the saturated binding state, actinomycin D occupies all the base pairs of DNA as described above. Therefore, there should be no free base pairs and an inhibitory effect on the binding of the ruthenium complex should occur. But Figure 2
As the result of No. 2 shows, no increase in luminescence due to binding inhibition is observed at all. Therefore, it is considered that such a case does not occur, or that actinomycin D hardly inhibits the binding of the ruthenium complex even when bound to DNA. That is, it is considered that the ruthenium complex can bind to the major groove on the back side of the minor groove to which actinomycin D binds. Also, actinomycin D, unlike the ruthenium complex, strongly binds to the GC sequence of DNA, which is considered to be due to the fact that the binding inhibition of the ruthenium complex does not occur. Join).

A description will be given of the operation after the saturation bond ratio. Actinomycin D added beyond the saturation bond is in a free state as shown in FIG. However, as can be seen from the drug-luminescence curve (FIG. 22), the luminescence increased after the saturation binding, indicating that the binding of the ruthenium complex to DNA was inhibited. Muller and Crothers et al. Have reported that when a DNA binds to actinomycin D, the DNA strand and actinomycin D form a hydrogen bond at an early stage. With this in mind, actinomycin D, which was added after the saturation bond and could not bind to DNA, becomes
It is likely that it exists around the NA molecule and inhibits the binding of the ruthenium complex.

In summary, no increase in luminescence was observed in the presence of DNA to which actinomycin D had been previously bound, unlike in the case of cisplatin or daunomycin. This is because, since the binding mode of actinomycin D is a minor group bond, the ruthenium complex having a major group binding is not inhibited by actinonomycin D binding, and even though actinomycin D is already bound, the ruthenium complex is further increased. It may mean that the complex can bind to DNA.

[0091] The results of light emission measured when allowed to coexist chromomycin A ₃ chromomycin A ₃ (Chromomycin A ₃₎ ruthenium complex in advance was allowed to act DNA are shown in Figure 24. Chromomycin A ₃
Is a chromomycinone ring as shown in the figure and a total of 5
It is an antibiotic consisting of two sugar chains (hexapyranose). It binds to the GC (guanine / cytosine) sequence of DNA and inhibits RNA synthesis, but its DNA synthesis inhibitory activity is small. Recent studies have revealed the binding mode as follows. Chromomycin A ₃ binds to a DNA chain in the presence of a divalent metal ion such as magnesium ion (Mg ²⁺ ) in a molar ratio of chromomycin A ₃ : Mg ²⁺ : DNA chain = 2: 1: 1. . At that time, two chromomycin A ₃ molecule either in the minor groove of DNA strands, or 2 molecules in line with it is attached in line at point symmetry. At this time, the number of base pairs required for binding is chromomycin A.
₃ two molecules are said to be about 6 base pairs. Note that D
The amount of NA is 14.9 μM in terms of base pairs.

[0092] the binding mode of chromomycin A ₃ can be seen are many similarities with the binding mode of the actinomycin D.
Nucleotide sequence specificity in binding, and the like that inhibit RNA synthesis is similar points, bound to most importantly actinomycin D, chromomycin A ₃ both the minor groove of DNA, major groove is empty Is a point. If the above "actinomycin D" last minor groove binding drugs as described in the section can not inhibit the binding of DNA ruthenium complex, as with actinomycin D in the case of this chromomycin A ₃ It is considered that the light-emitting characteristics described above can be obtained.

The results of the light emission characteristics and the saturation coupling were as follows. Graph in drug amount-luminescence curve
(DNA (-)) than, large change in light emission amount even amount of drug when coexist only chromomycin A ₃ increases was observed. This chromomycin A ₃ itself represents that does not have a significant effect on the electrochemiluminescence of the ruthenium complexes. On the other hand, from the graph shown on the left (DNA (+)), an increase in light emission amount with increasing amount of drug that acts on DNA such as cisplatin and daunomycin are in the presence of reacted beforehand chromomycin A ₃ DNA observed Was not done.
However, when the amount of chromomycin A ₃ was excessive, the amount of luminescence increased.

[0094] then calculated saturation binding ratio of chromomycin A ₃ and DNA. FIG. 25 shows the result. The format of the graph is the same as that for daunomycin in FIG. The relative amounts of chromomycin A ₃ to the DNA in a saturated bond ratio was 0.35. That is, chromomycin A ₃ is D
It was found that one molecule binds to NA 2.85 base pairs. Chromomycin A ₃ occupies about 6 base pairs in 2 molecules, so 1
It is about 3 base pairs per molecule. Thus as in the case of actinomycin D is a saturated bond state, chromomycin A ₃ will be occupying almost all base pairs of DNA. This saturation binding ratio was applied to the drug amount-emission curve of FIG. 24 and represented by a chain line.

[0095] emission characteristics of chromomycin A ₃ As is apparent from the above results was a good very homologous with actinomycin D. This suggested that in the case of a minor groove-binding drug, the binding of the ruthenium complex to DNA did not occur, and no increase in luminescence was observed.

The above is summarized as follows. Four anti-cancer and anti-viral agents, namely cisplatin,
Daunomycin, actinomycin D, consider electrochemiluminescent properties of the ruthenium complex with DNA presence for chromomycin A _3, were discussed each binding mode. As a result, the following findings were obtained.

Cisplatin binds to or intercalates with major grooves in DNA, and that binding has been shown to occur more than once every two base pairs without obeying the neighborhood exclusion rule. Also, by binding to a major groove, the compound exhibits a binding inhibiting effect of a ruthenium complex.

Daunomycin is the ruthenium complex D
Since it inhibits the binding to NA with high efficiency, it binds to the AT site having good conjugation with the ruthenium complex. At this time, it is concluded that the presence of daunosamine in the major groove enhances the binding inhibitory effect.

In the case of DNA to which actinomycin D is bound in advance, unlike the case of cisplatin or daunomycin, the binding of the ruthenium complex is not inhibited. This is because, since the binding mode of actinomycin D is a minor groove bond, the ruthenium complex having major groove binding is not inhibited by actinomycin D binding, and even though actinomycin D is already bound, the ruthenium complex is further increased. It indicates that the complex can bind to DNA.

[0100] emission characteristics of chromomycin A ₃ that exhibit very similar binding modes and actinomycin D was a good thing very homology with actinomycin D. This indicates that, in the case of a drug having a minor groove binding, no increase in luminescence is observed because the binding of the ruthenium complex to DNA is not inhibited.

Considering the above, it is possible to estimate whether the binding position of the DNA drug is a minor groove or a major groove by changing the electrochemiluminescence characteristics of the ruthenium complex. FIG. 26 shows this. That is, when a part or the whole of the drug is present in the major groove, the binding of the ruthenium complex is inhibited, and an increase in light emission is observed. When the drug is present in the minor groove, such binding inhibition does not occur, and substantially no increase in luminescence is observed.

Evaluation of this Method in a State Closer to the In Vivo System The environment in which the anticancer agents and antiviral agents such as cisplatin and actinomycin D taken up in this example actually exert their pharmacological effects is naturally in vivo. However, the evaluation in this example is performed in vitro. Therefore, the validity of this method is i
We decided to evaluate it in the presence of histones as a state closer to the n vivo system. Table 4 shows the results.

[0103]

[Table 4]

The amount of luminescence was evaluated as a relative value with 100 being the case. The light emission amount at (70.0) is the light emission amount at (65.
9) It turns out that it is bigger. Is the luminescence in the presence of the DNA with the histone, and is the luminescence in the case of the coexistence of the DNA alone. This means that histones are DNA
Shows that the binding of the ruthenium complex to DNA is inhibited by binding to. It is an expected phenomenon that the binding of the complex is inhibited by the binding of a large molecule such as a histone to DNA as compared with the ruthenium complex.
A is all covered with histone. Therefore, the ruthenium complex can be converted into DNA by histone without adding a drug.
Binding is completely inhibited. Therefore, it can be seen that histone binding is not always a convenient factor for the evaluation of the present method. However, in the presence of the low-concentration histone used in this study, the binding inhibition was small, and it seems to be sufficient for drug evaluation. When compared with, the light emission amount is significantly larger than. In and, only less than half the saturation amount of cisplatin is bound to DNA.
Therefore, the binding inhibitory effect of cisplatin should be small and the increase in luminescence should be small. Nevertheless, when histones coexist, luminescence is significantly increased. This indicates that the synergistic effect of the inhibition of ruthenium complex binding by histone and that by cisplatin resulted in the inhibition of binding exceeding the amount of cisplatin added. Considering the above, this method indicates that the inhibitory effect of the drug itself and the inhibitory effect of the protein cannot be distinguished in the presence of a DNA-binding protein such as histone. For this reason, this method uses DN
It seems appropriate to consider this as an evaluation method in an in vitro system in which binding between the A molecule and the drug occurs directly.

In this example, a state close to in vivo is obtained by adding a histone to the system. However, phage-derived lambda DNA and bovine thymus-derived histones are accurately expressed in vivo.
It is not clear whether or not the chromatin structure of DNA can be taken. On the other hand, DN on which anticancer drugs act
A may not be DNA having a chromatin structure, but may be "naked DNA" that is not bound, such as histone. In other words, cells such as cancer cells and bone marrow cells, in which drugs such as anticancer drugs mainly exert their effects, are very active cells even in the living body, so that transcription and replication of DNA are actively performed. Therefore, a considerable amount of the DNA of such cells does not have a chromatin structure, and actively performs transcription and replication reactions as DNA molecules, and drugs such as anticancer drugs act on “naked DNA”. As a result, it is considered that the medicinal effect mainly appears in such active cells.

[0106]

In this embodiment as EFFECT above, Ru (phen) ₃ ²⁺ cisplatin as electrochemiluminescence probe, daunomycin,
Actinomycin D, 4-drug of chromomycin A ₃ was applied for the purpose of evaluating the binding mode of the DNA. As a result, the electrochemiluminescence characteristics of the ruthenium complex in the presence of DNA in which these drugs have been acted in advance reflect the binding mode of each drug. That is, the binding mode of each drug can be classified by using the drug amount-luminescence curve. Specifically, when the curve of the amount of drug-luminescence becomes an upward-sloping graph, it indicates that the drug is arranged in the major groove of DNA, and when the luminescence curve is substantially parallel to the horizontal axis, the drug is measured. Indicates that the DNA is arranged in a portion other than the major groove (minor group) of the DNA.

Further, when this method was evaluated in a state closer to the in vivo system (in the presence of histone), the inhibition of binding of the ruthenium complex to DNA by the histone and the drug occurred simultaneously. The results suggest that it would be difficult,
Appropriate for application to in vitro systems.

The results obtained as a whole are summarized as follows.

A ruthenium complex using a phenanthroline compound such as Ru (phen) ₃ ²⁺ or a bipyridine as a ligand is
Electrochemiluminescence was performed, and in the presence of DNA, the luminescence of the ruthenium complex decreased as the amount of DNA increased. Thus electrochemiluminescence such Ru (phen) ₃ ²⁺ to interact with DNA can be utilized as a probe to drugs that act on DNA.

When Ru (phen) ₃ ²⁺ or the like was applied to a DNA-acting drug as an electrochemiluminescent probe, the electrochemiluminescent properties of the ruthenium complex in the presence of DNA in which the drug had previously acted were determined for each drug. Changes to reflect the binding mode of

By utilizing a drug amount-luminescence curve in the presence of DNA in which a drug has been acted on beforehand,
The binding mode of each drug can be classified. In other words, when the curve of the amount of drug-emission curve rises to the right, it is determined that the drug is located in the major groove of the DNA. When the emission curve is substantially parallel to the horizontal axis, the drug is DN.
Part other than the major groove of A (minor groove)
Is determined.

[0112] From the above results, by using the Ru (phen) ₃ ²⁺ ruthenium complexes such as electrochemiluminescence probe, anticancer agents that act on DNA, various information for binding mode of antiviral agents obtained It became clear that it could be done.

The electrochemiluminescent probe used in this example is Ru
is a (phen) ₃ ^2+, complexes of ruthenium are known various ones not limited thereto. Some of them are Ru (phen) ₃
Some have a different effect than ²⁺ . For example, the ligand can be changed from phenanthroline to diphenylphenanthroline.
Ru converted to (DIP) (DIP) ₃ ²⁺ is, Ru (phen) ₃ D than ²⁺
Strong binding to NA.

Those acting on DNA include not only the above-mentioned drugs but also many biological substances such as DNA-binding proteins. Such a substance exerts a great influence on a biological reaction by performing various actions on DNA controlling many biological information. By using this method, it is possible to determine whether the binding position of a drug acting on DNA is on the major groove side or the minor groove side. These findings are important in elucidating the mechanism of action of proteins. Therefore, this method is called DN
When applied to the A-binding protein, it becomes possible to expand the application range not only to drugs but also to various proteins.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing a binding mode when a Δ-form of two enantiomers of a ruthenium phenanthroline complex is bound.

FIG. 2 is a diagram showing an evaluation principle of screening for a DNA-binding drug.

FIG. 3 shows the chemical structures of various drugs.

FIG. 4 is a graph showing the electrochemiluminescence characteristics of a ruthenium complex in the presence of λDNA.

FIG. 5 is a diagram showing a principle of measuring a binding mode of a drug based on a relationship between an amount of a complex in a free state and an amount of electrochemiluminescence.

FIG. 6 is a schematic diagram of a measuring device.

FIG. 7 is a schematic diagram of a potential scan and a light emission pattern.

FIG. 8 is a diagram showing potential dependence of light emission in a system to which sodium oxalate is not added.

FIG. 9 is a diagram showing potential dependence of luminescence in a system to which sodium oxalate (15 mM) is added.

FIG. 10 is a diagram showing a step potential and a light emission pattern in the absence of oxalate.

FIG. 11 is a diagram showing a potential change and a light emission pattern in the presence of oxalate (15 mM).

FIG. 12 is a diagram showing a luminescence pattern in the presence of oxalate and DNA.

FIG. 13 is a graph showing the dependence of light emission in the absence of oxalate on the concentration of a ruthenium complex.

FIG. 14 is a graph showing the dependence of luminescence in the presence of oxalate (15 mM) on the concentration of a ruthenium complex.

FIG. 15 is a schematic diagram showing a method for measuring a saturation binding ratio between DNA and a drug.

FIG. 16 is a diagram showing a cisplatin amount-luminescence curve and a structural formula of cisplatin.

FIG. 17 is a diagram showing two binding modes of cisplatin and DNA.

FIG. 18 is a graph showing a measured luminescence when a ruthenium complex is allowed to coexist with DNA on which daunomycin has been acted.

FIG. 19 is a graph showing a saturation binding ratio between daunomycin and DNA.

FIG. 20 is a diagram showing the state of binding of daunomycin to DNA.

FIG. 21 is a diagram showing a state after correction of a measured luminescence amount.

FIG. 22 is a graph showing measured luminescence when a ruthenium complex is allowed to coexist with DNA on which actinomycin D has been acted.

FIG. 23 is a diagram showing a saturation binding ratio between actinomycin D and DNA.

24 is a diagram showing a light emission measurement amount when allowed to coexist ruthenium complex in DNA is reacted with chromomycin A _3.

25 is a diagram showing a saturation binding ratio of chromomycin A ₃ and DNA.

FIG. 26 is a diagram showing the binding position of a drug acting on DNA.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Dark box 2 Photomultiplier tube 3 Power supply 4 Emission measurement part 5 Potentiiostat 6 Function generator 7 Oscilloscope 8 Recording part 9 Electrochemical cell

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────────────────────────────────────────────────── ─── Continuing from the front page Application for Article 30 (1) of the Patent Act has been made. Published on March 19, 1992, published in the Electrochemical Society of Japan 59th Annual Meeting Abstracts, p.262. (56) References JP-A-2-11597 (JP, A) JP-A-2-72900 (JP, A) (58) Fields studied (Int. Cl. ⁷ , DB name) G01N 21/76 C12Q 1 / 68 G01N 33/15 G01N 33/50 BIOSIS (DIALOG) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A method for determining the mode of binding of a DNA agent in a sample solution to DNA, wherein the ruthenium complex is dispersed in the sample solution, and a current is applied to the ruthenium complex to cause electrochemical emission. A method for identifying the binding mode of a DNA agent, wherein the mode of binding of the DNA agent to DNA is identified based on the state of light emission. 2. When the amount of luminescence increases due to an increase in the amount of a DNA agent, it is determined that the DNA agent is bound to a major groove of DNA, while when the amount of luminescence does not substantially increase, the major 2. The method for identifying a binding mode of a DNA active substance according to claim 1, wherein the binding mode is determined to be binding to other than the groove. 3. The method according to claim 1, wherein a ruthenium complex having a phenanthroline compound as a ligand is used as the ruthenium complex. 4. A B-type DNA is used as the DNA,
4. The method for identifying a binding mode of a DNA agent according to any one of claims 1 to 3, which utilizes the fact that a Δ-form of a ruthenium complex binds to the B-type DNA. 5. The method according to claim 1, wherein an oxalate is present in the sample solution. 6. The method according to claim 1, wherein a potential step method is used as the current application method.
A method for identifying the binding mode of an NA agonist.