JP2003034600A - Growing method for large-size single crystal of dna oligomer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a growing method for a large-size single crystal of decamer DNA oligomers, to provide a growing method for a large-size single crystal of a decamer DNA oligomers that is usable for the analysis by a crystal neutron diffraction method, and to draw a phase diagram with respect to the crystallization of the decamer of DNA oligomers for growth of the large-size crystal of the decamer that has a size of >=1 mm square. SOLUTION: In the growing method for a large-size single crystal of the decamer DNA oligomers, a single crystal of decamer DNA oligomers is deposited in a batch process under the conditions that the concentrations of DNA and MgCl2 are in the region upper than each curved line shown in Fig. 1, in the presence of 20-30% 2-methyl-2,4-pentanediol(MPD).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for growing large single crystals of decamer DNA oligomers. More specifically, the present invention relates to making a phase diagram of crystallization of a 10-mer DNA oligomer and making a large single crystal of the 10-mer DNA oligomer based on this phase diagram.

[0002]

2. Description of the Related Art Many studies have been conducted on the roles of water molecules and hydrogen bonds involved in the structure and function of DNA. For example, water molecules are DNA-binding proteins and DNA
It plays a role like "glue" that connects with (Ot
winowski, Z., et al., Nature, 335 (1988) 321-32
9), and that water molecules are regularly arranged in the minor groove of the B-type DNA double strand and contribute to the structural stabilization (Denisov, VP et al., J. Mol. Biol., 268 (19
97) 118-136) are known. From such studies, there is a network of water of hydration around nucleic acid molecules,
It is thought to be closely related to the recognition of genomic information (Otwinowski, Z., et al., Nature, 335 (1988)
321-329). It is also considered that the base pair of DNA influences the coordination of water of hydration in the network, which may be recognized by the protein (Otwinowski, Z., et a.
L., Nature, 335 (1988) 321-329).

Conventionally, X-ray diffraction, which has been mainly used for structural analysis of DNA, has been able to confirm the positional information of oxygen atoms of hydrated water in DNA. However, whether or not the base pair of DNA influences the coordination of hydration water in the hydration water network around the nucleic acid molecule based on the positional information of such oxygen atom alone, the protein determines whether hydration water in the DNA is present. It is not possible to confirm whether or not the coordination of is recognized. Thus, it is important to completely determine the hydration structure containing hydrogen in order to accurately determine the coordination of water of hydration in DNA. Not not. For that purpose, it is necessary to analyze the DNA molecule by the neutron crystal diffraction method.

Currently, the highest performance neutron diffractometers (eg, BIX-
When 3) is used, there is a problem that a very large crystal must be used as a sample because of problems such as available beam intensity. For example, chicken egg white lysozyme (Niimura, N. et al., Nat. Struct. Biol.,
11 (1997) 909-914), rubredoxin, myoglobin, and other neutron crystal diffraction experiments of proteins all required crystals with a size of several mm square.

The present inventors have used a DNA crystal of 0.3 mm ³ or less in a DNA structural analysis experiment using a neutron crystal diffraction experiment, but could not obtain a sufficient analysis result. Therefore, in the neutron crystal diffraction experiment of DNA, it is considered necessary to use a DNA crystal with a size larger than this, and considering the case of the protein described above, it is expected that a crystal with a size of 1 mm square or more is required. To be done. However, no large single crystal of such DNA has been obtained so far.

[0006] The salt concentration gradient method using a capillary, which has been used for crystallization of biomolecules such as proteins and nucleic acids until now, puts a metal salt powder at the bottom of the capillary,
Inject a mixed solution containing biomolecules and precipitants on top of it,
This is a method of crystallizing a biomolecule. In this method, the metal salt at the bottom of the capillary diffuses into the mixed solution and forms a salt concentration gradient by the action of gravity (Ataka,
M., et al., JAERI-M, 92-213, 61). We have
By applying this crystallization method to DNA, it is possible to precipitate DNA crystals in this salt concentration gradient within a range of salt concentrations that is suitable for precipitating DNA in relation to DNA concentration. I tried.

According to this method, since DNA molecules can be continuously supplied from the vicinity of the crystal deposition position, it becomes possible to obtain a crystal larger than before. However, in reality, the crystals of DNA have a high specific gravity, so that the crystals formed in the capillaries precipitate at the bottom of the capillaries with the passage of time, and large crystals cannot be obtained. Therefore, in order to achieve efficient crystallization of DNA, it is necessary to prepare a concrete phase diagram of DNA crystallization.

To date, several studies have been conducted on the phase diagram for DNA crystallization. For example, create a phase diagram for DNA and spermine concentrations to study spermine and Mg ²⁺ ion binding constants for crystallization of tetrameric, hexameric, heptameric, octameric, and heptameric DNA. Study (Ma
linina, LV et al., J. Biomol. Struct. Dyn., 5 (1
987) 405-433), a phase diagram for the crystallization of hexamer DNA (d (CGCGCG)), and showed that the crystal system differs depending on the crystallization conditions (Malinina, LV etal., J.
Cryst. Growth, 110 (1991) 252; Minasov, G. et a
l., J. Cryst. Growth, 122 (1992) 136), and prepared a phase diagram of the concentration of 2-methyl-2,4-pentanediol (MPD) as a precipitant and the concentration of inorganic cations to obtain hexamer DNA.
(D (CICICI), where I represents inosinic acid) showed that crystals could be obtained with both B-type DNA and Z-type DNA depending on the crystallization conditions (Ho, PS et al., Science,
254 (1991) 1003-1006) and the like are known.

However, the previous studies have not reported at all on the phase diagram in the presence of a high-concentration inorganic cation using a 10-mer DNA oligomer.
Moreover, in order to make the hydration structure of the DNA double strand closer to the in vivo condition, it is considered necessary to have a structure with one or more pitches of the helix. In addition, various crystallization methods have been used in previous studies on phase diagrams,
It was difficult to interpret the crystal growth process of double-helix DNA uniformly.

[0010]

[Problems to be Solved by the Invention]
The objective is to develop a method for growing large single crystals of oligomers. Another object of the present invention is to develop a method for growing a large single crystal of a 10-mer DNA oligomer, which is suitable for analysis by neutron crystal diffraction method. Another object of the present invention is to prepare a phase diagram of crystallization of a 10-mer DNA oligomer in order to grow a large single crystal of a 10-mer DNA oligomer having a size of 1 mm square or more.

[0011]

The present invention provides 20-30% of 2-
In the presence of methyl-2,4-pentanediol (MPD), by a batch method under the conditions of DNA concentration (mM) and MgCl ₂ concentration (mM) located in the region above each curve shown in FIG. The problem is solved by providing a method for growing a large single crystal of a 10-mer DNA oligomer, which comprises depositing a single crystal of a 10-mer DNA oligomer.

As used herein, the "batch method"
The concept is a concept that includes both the large batch method and the microbatch method.Specifically, a biomolecule, a precipitating agent, etc. are mixed in a solution to a target concentration, and the mixture is kept under constant temperature conditions. This is done by letting it stand. The batch method is capable of precisely adjusting the concentration condition of the solution, as compared with a conventional method such as a vapor diffusion method.
Large-scale DN that has never existed because it is easy to enlarge the experimental system
A crystal can be obtained.

The DNA oligomer used in the present invention for crystallization by the batch method is characterized in that it is a nucleotide having a length of one spiral or more. This is because the hydration structure of the DNA double strand is brought closer to the in vivo condition for discussion. One helix pitch of DNA is 10.7 nucleotides for A-type DNA and 10.0 for B-type DNA.
Nucleotides, 12 nucleotides for Z-type DNA. Therefore, the length of the DNA oligomer is 11 in the case of A-type DNA.
It must be a DNA oligomer having a nucleotide length of nucleotides or more, 10 nucleotides or more for B-type DNA, 12 nucleotides or more for Z-type DNA. In the present invention, 10
We focused our attention on the DNA of the polymer and advanced our research.

The DNA oligomer used in the present invention is 0.45.
When the concentration is lower than mM, the DNA oligomer in the solution does not precipitate regardless of how the other conditions are changed. Therefore, use a DNA oligomer concentration of at least 0.45 mM. Furthermore, the DNA oligomer used in the present invention is
If the concentration exceeds 5 mM, it cannot be dissolved in the solution, and as a result, it is not suitable for the method for growing a large single crystal of a DNA oligomer of the present invention. Therefore, the DNA oligomer used in the present invention is used in a concentration range of 0.45 to 5.0 mM or less.

In the method of the present invention, an inorganic cation is used as one of the crystallization agents. Specific examples of inorganic cations include, but are not limited to, MgCl ₂ , NaCl, KCl, NiCl ₂ . For crystallization of DNA oligomers it is preferred to use MgCl ₂ .

In the method of the present invention, a precipitating agent is used to precipitate the DNA more efficiently. Specific examples of the precipitant include, but are not limited to, organic solvents such as MPD, polyethylene glycol, and inorganic salts. For the crystallization of DNA oligomers, it is preferable to use MPD, which is commonly and conventionally used. Note that there is no strict definition that distinguishes between crystallization agents and precipitation agents.

In consideration of the above conditions, in the present invention, first, in order to find a suitable condition for crystallization of a decameric DNA oligomer, various concentrations of 20 to 30% as a precipitating agent are used. Crystallization phase diagrams are generated by the microbatch method using solutions containing various concentrations of decameric DNA oligomers up to 5 mM and various concentrations of MgCl ₂ as inorganic cations in the presence of MPD. To create a more detailed phase diagram, use MP
It is necessary to perform crystallization experiments with as many combinations of D concentration, decamer DNA oligomer concentration, and MgCl ₂ concentration.

In one embodiment of the present invention, the crystallization of the 10-mer DNA oligomer in various combinations of MPD concentration, 10-mer DNA oligomer concentration, and MgCl ₂ concentration results in a 10-mer DNA oligomer phase. A diagram can be created and included in the crystallized region of the phase diagram according to the phase diagram
Crystallization of decamer DNA can be performed under the conditions of DNA concentration, MPD concentration, and MgCl ₂ concentration.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION Selection of Sample In the present invention, first, a DNA oligomer used for preparing a phase diagram was selected. This DNA oligomer is B
It is type DNA. The base sequence of this DNA oligomer is Nuclei
c Acid Data Base (NDB), Protein Data Bank (PD
B), Biological Macromolecule Crystallization Data
Of the DNA oligomers contained in databases such as base (BMCD), 21 types of B-forms for which X-ray crystal structure analysis has been performed
I picked out the DNA. Their specific sequences and their physicochemical characteristics are shown in Table 1 below.

[0020]

[Table 1]

In the present invention, any of these sequences is subjected to a crystallization experiment by the hanging drop vapor diffusion method well known in the art to determine which
We searched whether the DNA sequence was suitable for crystallization. As the crystallization conditions, the conditions described in each paper and the nucleic acid crystallization condition retrieval kit (manufactured by Hampton Research) were used. As a result, a 10-mer DN having the nucleotide sequence shown in SEQ ID NO: 4
A oligomer, d (CCATTAATGG) is relatively large in a short time
Since it was found to form a DNA single crystal, this 10-mer DNA oligomer (SEQ ID NO: 4) was used in the following experiments.
Was used.

Preparation of Phase Diagram by Microbatch Method In order to prepare a phase diagram, the microbatch method was used in the present invention. In the vapor diffusion method described above, the water content in the mixed solution containing the DNA oligomer evaporates, so the DNA concentration and M
This is because the experimental conditions such as the concentration of PD change with time and are not suitable for clarifying the detailed relationship between crystallization and those experimental conditions. Further, in the vapor diffusion method, there is a limit to the enlargement of the experimental system for producing large crystals.

In the microbatch method, various concentrations of DNA oligomers, various concentrations of MgCl ₂ , and various concentrations of MPD in a buffer (100 mM sodium cacodylate, pH 7.0) were used.
4 μl of a mixed solution containing the above was added as a crystal mother liquor into a 96-well plate for crystallization, and paraffin oil was overlaid and sealed. This mother liquor was allowed to stand at 6 ° C. to crystallize the DNA in the mother liquor.

The concentrations of the DNA oligomer, MgCl ₂ and MPD used for crystallizing the DNA are shown in Tables 2 to 5. Tables 2 to 5 also show the results of whether or not crystals were precipitated 20 days after the start of the experiment as a result of crystallization of DNA under each condition. Table 2 shows MPD of 30% (v / v), Table 3 shows MPD of 25% (v / v), and Table 4 shows MPD of 22.5.
% (V / v), Table 5 shows the experimental conditions and data for MPD of 20% (v / v).

[0025]

[Table 2]

[0026]

[Table 3]

[0027]

[Table 4]

[0028]

[Table 5]

The results of DNA single crystal precipitation under these conditions are shown as a graph in FIG. In Figure 1, MPD
When the concentration is 20%, 22.5%, 25%, and 30%,
It is shown that the DNA oligomer having the sequence of CCATTAATGG (SEQ ID NO: 4) crystallizes at DNA and MgCl ₂ concentrations in the area above the curve. From this phase diagram,
The crystal precipitation boundary line of the 10-mer DNA oligomer is
Even in terms of concentration, salt concentration (ie Mg in the present invention)
It was found that the boundary line decreased as the salt concentration increased on the relatively low Cl ₂ concentration side, and increased again as the salt concentration increased. like this
The characteristics of the phase diagram of DNA oligomers were investigated by Malinina et al. (Mal
inina, LV et al., J. Biomol. Struct. Dyn., 5 (19
87) 405-33) and Ho et al. (Ho, PS et al., Scienc
e, 254 (1991) 1003-1006).

Therefore, the present invention is based on the above results, that 20-30% of 2-methyl-2,4-pentanediol (M
In the presence of PD), single crystals of 10-mer DNA oligomers were prepared by the batch method under the conditions of DNA concentration (mM) and MgCl ₂ concentration (mM) located in the region above each curve shown in FIG. 1. A new method for growing a large single crystal of a 10-mer DNA oligomer is provided, which is characterized by precipitation.

When the details of crystallization of the 10-mer DNA oligomer are examined, as the MPD concentration is increased from 20%, the regions of DNA concentration (mM) and MgCl ₂ concentration (mM) in which crystals are precipitated are expanded. Furthermore, at each MPD concentration, DNA concentration (mM) and MgCl ₂ concentration (mM) near the crystal precipitation boundary line
Comparing single crystals of decameric DNA obtained under the conditions
There was a tendency that the outer shape of the crystal was adjusted as the concentration increased. It is thought that this is because MPD, which is an organic solvent, lowers the dielectric constant of the solution, and as a result, electrostatic interaction is strengthened and DNA molecules are easily associated.

In FIG. 1, it is shown that, in the crystal precipitation region, the crystal precipitation boundary line of DNA draws a downward convex curve regardless of the concentration range of MPD of 20 to 30%. .
Although previous studies have shown that the size of the crystal precipitation region depends on the MPD concentration (Minasov,
G. et al., J. Cryst. Growth, 122 (1992) 136), DN
Nothing was known about the minimal solubility of A. From this result, DN in the crystal precipitation region
It is suggested that the property that the crystal precipitation boundary line of A draws a downward convex curve is not a property that depends on the alcohol concentration but a property that appears specifically in the presence of an inorganic cation, especially MgCl _2. .

The above-mentioned results are shown in MPD concentration of 20%, 22.5%, 25
%, And the respective data when set to 30% will be described in more detail. Figure 2 shows the phase diagram when the MPD concentration is 30%. It is shown that single crystals of decamer DNA oligomers can be deposited by the batch method under conditions of DNA concentration (mM) and MgCl ₂ concentration (mM) located in the region above the curve shown in this figure. There is. As can be seen from this figure, under the condition that MgCl ₂ does not exist, crystals of the 10-mer DNA oligomer do not precipitate at any concentration of the DNA oligomer, but when the MgCl ₂ concentration is 7.5 mM, It was found that at a DNA concentration of 4.5 mM, crystals of decamer DNA oligomers were precipitated. Also, the MgCl ₂ concentration is 600 mM.
In the case of, the crystals of 10-mer DNA oligomers were precipitated at a DNA concentration of 3.0 mM or 4.0 mM, whereas MgC
10-mer DN at l ₂ concentrations of 700 mM or higher
It was found that crystals of A oligomer did not precipitate. From this, in a preferred embodiment of the present invention, the 10-mer
For the precipitation of DNA oligomer single crystals, MgCl ₂ at a concentration of 7.5 mM to 600 mM is used at a MPD concentration of 30%.

In a detailed embodiment of the invention, 30% MP
In the presence of D, at a DNA concentration of 5.0 mM or less,
When creating a graph with MgCl ₂ concentration (mM) as the X-axis and DNA concentration (mM) as the Y-axis, the following (MgCl ₂ concentration, DNA
Concentration); (7.5, 5.0); (7.5, 4.5);
(15, 1.8); (50, 0.9); (200, 0.9); (500, 1.
8); (600, 3.0); and (600, 5.0); the DNA concentration (mM) contained in the region where Y is larger than the polygonal line formed by connecting the points in this order with a straight line, and
Batches of 10-mer DNA at MgCl ₂ concentration (mM)
Provided is a method for growing a large single crystal of a 10-mer DNA oligomer, which comprises depositing a single crystal of an oligomer.

A phase diagram when the MPD concentration is 25% is shown in FIG. It shows that single crystals of DNA oligomers can be deposited by the batch method under conditions of DNA concentration (mM) and MgCl ₂ concentration (mM) located in the region above the curve shown in this figure. As can be seen from Fig. 2, MgCl ₂
In the absence of A, crystals of 10-mer DNA oligomers did not precipitate at any concentration of DNA oligomer, but 4.0 mM and 2.5 mM at a MgCl ₂ concentration of 50 mM.
It was found that crystals of 10-mer DNA oligomers were precipitated at the DNA concentrations of. It was also found that when the MgCl ₂ concentration was 600 mM, crystals of the decamer DNA oligomer were precipitated at a DNA concentration of 4.5 mM. From this, in a preferred embodiment of the present invention, in order to carry out precipitation of a 10-mer DNA oligomer single crystal, at a MPD concentration of 25%, 50 mM
Use MgCl ₂ at a concentration of from 600 mM.

In another detailed aspect of the invention, 25%
In the presence of MPD, the concentration of MgCl ₂ (mM) is on the X-axis and the concentration of DNA (mM) is Y at 5.0 mM or less.
When creating a graph with axes, the following combinations of (MgCl ₂ concentration, DNA concentration); (50, 5.0); (50, 2.
5); (200, 1.0); (300, 1.5); (600, 4.5); and (600, 5.0); It is characterized by precipitating a single crystal of a 10-mer DNA oligomer by a batch method at a DNA concentration (mM) and a MgCl ₂ concentration (mM) contained in a large region. Provide a training method.

A phase diagram when the MPD concentration is 22.5% is shown in FIG. DNA located in the area above the curve shown in this figure
It is shown that under the conditions of concentration (mM) and MgCl ₂ concentration (mM), it is possible to deposit a single crystal of a 10-mer DNA oligomer by a batch method. As can be seen from this figure, under the condition where MgCl ₂ is not present, what concentration of D
Crystals of 10-mer DNA oligomers did not precipitate even when NA oligomer was used, but when MgCl ₂ concentration was 100 mM, D of 4.5 mM was obtained.
It was found that crystals of 10-mer DNA oligomers were precipitated at NA concentration. When the MgCl ₂ concentration is 400 mM,
Crystals of decamer DNA oligomers were precipitated at a DNA concentration of 3.0 mM or 4.5 mM, whereas MgCl ₂ concentration was 600
It was found that the crystals of the decamer DNA oligomer did not precipitate at a concentration of mM or higher. From this, in a preferred embodiment of the present invention, MgCl ₂ at a concentration of 100 mM to 400 mM is used at a MPD concentration of 22.5% in order to carry out precipitation of a 10-mer DNA oligomer single crystal.

In a further detailed aspect of the invention,
At a DNA concentration of 5.0 mM or lower in the presence of 22.5% MPD, the MgCl ₂ concentration (mM) is used as the X-axis and the DNA concentration (m
If you create a graph with (M) as the Y-axis, the following (MgCl
₂ concentrations, DNA concentration); (100, 5.0); (10
(0, 4.5); (200, 3.0); (400, 3.0); and (40
0, 5.0); each of such points is connected by a straight line in this order, and at the DNA concentration (mM) and MgCl ₂ concentration (mM) contained in the region where Y is larger than the polygonal line, the batch method is used. The present invention provides a method for growing a large single crystal of 10-mer DNA oligomer, which comprises depositing a single crystal of 10-mer DNA oligomer.

A phase diagram when the MPD concentration is 20% is shown in FIG. It shows that single crystals of DNA oligomers can be deposited by the batch method under conditions of DNA concentration (mM) and MgCl ₂ concentration (mM) located in the region above the curve shown in this figure. As you can see from this figure, MgCl ₂
In the absence of H2O2, crystals of 10-mer DNA oligomers did not precipitate at any concentration of DNA oligomer, but when MgCl ₂ concentration was 200 mM, 10-mer DNA oligomer was obtained at 4.5 mM DNA concentration. It was found that crystals of oligomers were precipitated. Also, when the MgCl ₂ concentration is 400 mM, D of 4.5 mM
It was found that the crystals of the 10-mer DNA oligomer were precipitated at the NA concentration, whereas the crystals of the 10-mer DNA oligomer were not precipitated at the MgCl ₂ concentration of 600 mM or higher. From this, in a preferred embodiment of the present invention, in order to carry out precipitation of a 10-mer DNA oligomer single crystal, at a MPD concentration of 20%, 200 mM to 400 m
Use MgCl ₂ at a concentration of M.

In yet another detailed embodiment of the present invention, at a DNA concentration of 5.0 mM or less in the presence of 20% MPD, the MgCl ₂ concentration (mM) is on the X-axis and the DNA concentration (mM) is When creating a graph with the Y axis, (M
gCl ₂ concentration, DNA concentration); (200, 5.0);
(200, 4.5); (400, 4.5); and (400, 5.0); the DNA concentration in the region where Y is larger than the polygonal line created by connecting the points in this order with a straight line ( m
M) and MgCl ₂ concentration (mM) by the batch method,
Provided is a method for growing a large single crystal of a 10-mer DNA oligomer, which comprises depositing a single crystal of a 10-mer DNA oligomer.

In a more preferred embodiment of the present invention, 10
Provided is a method for growing a large single crystal of a 10-mer DNA oligomer, characterized in that the monomer DNA oligomer has a sequence of CCATTAATGG (SEQ ID NO: 4).

Examination of large-scale single crystal growth method In one embodiment of the present invention, various methods such as a salt concentration gradient method and a large batch method are used according to the above-mentioned crystallization conditions of a DNA oligomer to actually grow a large-sized single crystal. It was examined whether or not crystals were formed. For example, the salt gradient method puts MgCl ₂ at the bottom of the capillary and injects a mixed solution containing DNA and MPD, which is a precipitating agent, at the top of the capillary.
This is done by crystallizing the DNA. Specifically, when MgCl _{2 at} the bottom of the capillary diffuses into the mixed solution, a salt concentration gradient is formed by the action of gravity, and a salt concentration suitable for precipitating DNA in this salt concentration gradient is formed. It is performed by precipitating DNA crystals in the range of. The large batch method is performed by injecting a crystallization solution containing MgCl ₂ , DNA and MPD as a precipitating agent into a flat bottom plate, and leaving it at 6 ° C. for 2 to 3 months.

Preparation of DNA Large Single Crystal In one embodiment of the present invention, a DNA is prepared by the above-described crystallization method of a 10-mer DNA oligomer and has physicochemical properties suitable for structural and crystallographic analysis. Decamer DNA oligomer large single crystals are also provided. In a more preferred embodiment of the present invention there is provided a large single crystal of a decameric DNA oligomer having the sequence CCATTAATGG (SEQ ID NO: 4).

Here, the physicochemical properties suitable for the structural / crystallographic analysis are at least 0.2 × 0.2 × 0.2 mm.
³ , more preferably 0.5 × 0.5 × 0.5 mm ³ , most preferably
It has a size of about 1.0 × 1.0 × 1.0 mm ³ and can confirm the deflection of the crystal with a polarizing microscope, and it is possible to obtain clear diffraction spots by X-ray explanation experiments and neutron diffraction experiments. However, the present invention is not limited to these.

[0045]

EXAMPLES In order to understand the present invention in more detail, a 10-mer DN having the sequence of CCATTAATGG (SEQ ID NO: 4) is given below.
Examples for A oligomers are described. These examples are intended to be illustrative of the invention and not limiting of the scope of the invention.

Example 1: Examination of suitable conditions Based on the phase diagram of crystallization of a DNA oligomer having the sequence of CCATTAATGG (SEQ ID NO: 4) prepared by the present invention, MPD concentration, DNA oligomer concentration, and From the viewpoint of MgCl ₂ concentration,
The conditions suitable for crystallization of decamer DNA oligomer were investigated.

First, the MPD concentration will be examined. The MPD concentration has a characteristic that the crystal precipitation region expands as the concentration increases from 20% to 30%. This shows that in the case of MPD concentration between 20% and 30%, as the MPD concentration increases, even if the concentration of the DNA oligomer is lower, crystals are more likely to precipitate. ing. Therefore, in one embodiment of the present invention,
The MPD concentration was set to 30%.

Next, 30% MPD concentration conditions 20 days after the start of the experiment
DNA oligomer concentration based on the phase diagram below (Figure 2)
And MgCl₂Consider the relationship with concentration. 20 days after the start of the experiment
Is MgCl ₂In the vicinity of the crystal precipitation boundary line with a concentration of 200 mM or less,
It has been found that relatively large crystals can be obtained. This is
Noguchi, MgCl₂Crystallization occurs when the concentration is fixed
Use the lowest possible DNA oligomer concentration
Indicates that large crystals can be obtained. In particular,
2.7 mM of DNA oligomers and 1
5 mM MgCl₂1.0 × 1.0 × 0.1 mm under³degree
Relatively large crystals were obtained.

However, as can be seen from the crystal precipitation region of FIG. 2, under the condition that the MgCl ₂ concentration is 100 mM or less, the salt concentration increases and the crystal precipitation boundary line sharply drops and becomes very slight. From the results of the present invention, it was found that there is a difficulty in controlling the experimental conditions that the crystal precipitation is greatly affected by the difference in salt concentration. It was also found that this tendency becomes stronger as the concentration becomes lower. Although it is preferable to set conditions as close as possible to the crystal precipitation boundary line, it is difficult to control the MgCl ₂ concentration during the experiment.

Therefore, from the viewpoint of strictness or difficulty of control of experimental conditions, the conditions of 2.7 mM DNA oligomer and 15 mM MgCl ₂ described above were not used in the present invention. In the present invention, although it cannot be said that the conditions for the DNA oligomer concentration and the MgCl ₂ concentration are not optimal, it is easier to control the experimental conditions, in the presence of 1.5 mM DNA oligomer and 100 mM MgCl ₂ , 10 It was decided to crystallize the body DNA oligomer.

Reference Example: Crystallization Using Salt Concentration Gradient Method In the present invention, based on the above results, in the following of the present invention, the suitable conditions (30% MPD, 1.5 mM DN) described above were used.
A single crystal of DNA was prepared under A and 100 mM MgCl ₂ . Then, a salt concentration gradient method was used as a crystallization method of the DNA oligomer in this case. Crystallization was investigated using the salt gradient method because the salt gradient method has been used in the conventional production of protein crystals, and, for example, using chicken egg white lysozyme, 3.0 × 3.0 × 4.0
This is because it has been reported that a large crystal of mm ³ was created (Yoshiaki Minesaki, Ph.D., Japan Atomic Energy Research Institute, Advanced Science Research Center (2000)).

The salt concentration gradient method used for crystallization of DNA is such that MgCl ₂ is placed at the bottom of the capillary and DNA is placed above it.
It is a method of crystallizing DNA by injecting a mixed solution containing a precipitant and a precipitant. In the method of the present invention, the inner diameter is 2 mm and the length is 200 mm.
Add MgCl ₂ at the bottom of the capillary to 1.5 mM D
Inject in a mixed solution containing NA, 30% MPD, 100 mM sodium cacodylate. In this mixed salt concentration gradient, when MgCl ₂ diffuses to the combined solution, the action of gravity forms a salt concentration gradient. In this salt concentration gradient, DNA crystals are precipitated in a range of salt concentration suitable for precipitating DNA in relation to 1.5 mM DNA.

In the present invention, MgCl 2 is contained in the capillary.
_The amount of the crystallization solution was adjusted and injected so that ₂ could reach 100 mM in parallel, and the whole capillary was left standing at 6 ° C. As a result, extremely small crystals were deposited around the center of the capillary about 10 days after the start of the experiment. However, due to the high specific gravity of the crystals, those crystals were precipitated and aggregated in the capillaries over time, and large crystals were not formed. From this, it was found that the precipitation and aggregation of the crystals and the increase in the density act as an inhibiting factor for the formation of large-sized crystals of DNA.

Example 2: Crystallization using the large batch method According to the above-mentioned salt concentration gradient method and other general crystallization methods.
When we examined the reason why large single crystals of DNA oligomers could not be produced, it was found that not only the crystal growth is inhibited by chemical conditions such as salt concentration, but also the growth is inhibited by the physical aggregation of the crystals. It was also possible.

Therefore, in the present invention, a large-batch method was carried out using a flat-bottom plate so that the crystals were precipitated while being dispersed, and it was examined whether or not large crystals were formed.

Similar to the salt gradient method, 400 μl of a crystallization solution was prepared under conditions suitable for crystallization (30% MPD, 1.5 mM DNA, and 100 mM MgCl ₂ ). The crystallization solution was filtered using a membrane filter (MILLIPORE) and then poured into a flat-bottom VDX plate (Hampton Research) at 6 ° C.
Saved in.

Using a flat plate (VDX plate),
The crystallization solution was allowed to stand for 2-3 months, resulting in 1.7 × 1.3 × 0.6 mm
It was possible to make large single crystals of DNA oligomers having the sequence of CCATTAATGG (SEQ ID NO: 4) with a volume of ³ .

Example 3: Large size of the prepared DNA oligomer
Type single crystal CCATTAATGG (SE produced by the above-described method of the present invention
A single crystal of a DNA oligomer having a sequence of Q ID NO: 4) was analyzed by observation with an optical microscope and a method such as an X-ray crystal diffractometer.

CCAT prepared by the method of the present invention
Structural analysis of a single crystal of a DNA oligomer having the sequence of TAATGG (SEQ ID NO: 4) was performed by a neutron crystal diffraction experiment.

[0060]

INDUSTRIAL APPLICABILITY The present invention was able to prepare a phase diagram for its crystallization by using a decamer DNA oligomer.
Based on the phase diagram, 30% MPD, 1.5 mM DNA, and 100 mM Mg were used to crystallize the 10-mer DNA oligomer.
It was found that the conditions of Cl ₂ are suitable. Furthermore, in the present invention, under such conditions, by performing crystallization using a flat plate, 1.7 × 1.3 × 0.6 mm
^{3 with} a large volume that has never been obtained before 1
Large single crystals of 0-mer DNA oligomer could be prepared.

[0061]

[Sequence list] <110> Japan Atomic Energy Research Institute <120> Method of forming a large single crystal of DNA oligomer e crystal of DNA oligomer) <130> 011326 <160> 13 <200> 1 <211> 10 <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 1 ccaacgttgg 10 <200> 2 <211> 10 <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 2 ccagcgctgg 10 <200> 3 <211> 10 <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 3 ccaggcctgg 10 <200> 4 <211> 10 <212> DNA <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 4 ccattaatgg 10 <200> 5 <211> 10 <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 5 ccactagtgg 10 <200> 6 <211> 10 <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 6 ccaagcttgg 10 <200> 7 <211> 10 <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 7 cgatcgatcg 10 <200> 8 <211> 10 <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 8 cgattaatcg 10 <200> 9 <211> 10 <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 9 cgatatatcg 10 <200> 10 <211> 10 <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 10 catggccatg 10 <200> 11 <211> 10 <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 11 ccggcgccgg 10 <200> 12 <211> 10 <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 12 ctctcgagag 10 <200> 13 <211> 10 <212> DNA <213> Artificial sequence <220> <223> DNA oligomer for forming a single crystal of DNA. <400> 13 cgcaattgcg 10

[Brief description of drawings]

FIG. 1 shows CCATTAATGG (SEQ ID NO: under conditions of MPD concentrations of 20%, 22.5%, 25%, and 30%).
4 shows a phase diagram for crystallization of a DNA oligomer having the sequence of 4). It is shown that the DNA crystallizes at the DNA and MgCl ₂ concentrations present in the area above each curve.

[Fig. 2] Fig. 2 shows C under the condition of MPD concentration of 30%.
Figure 3 shows a phase diagram for crystallization of a DNA oligomer having the sequence CATTAATGG (SEQ ID NO: 4). At the concentrations of DNA and MgCl ₂ present in the area above each curve, DN
It shows that A crystallizes.

[Fig. 3] Fig. 3 shows C under the condition that the MPD concentration is 25%.
Figure 3 shows a phase diagram for crystallization of a DNA oligomer having the sequence CATTAATGG (SEQ ID NO: 4). At the concentrations of DNA and MgCl ₂ present in the area above each curve, DN
It shows that A crystallizes.

FIG. 4 shows a phase diagram for crystallization of a DNA oligomer having the sequence of CCATTAATGG (SEQ ID NO: 4) under the condition of MPD concentration of 22.5%. It is shown that the DNA crystallizes at the DNA and MgCl ₂ concentrations present in the area above each curve.

[Fig. 5] Fig. 5 shows C under the condition that the MPD concentration is 20%.
Figure 3 shows a phase diagram for crystallization of a DNA oligomer having the sequence CATTAATGG (SEQ ID NO: 4). At the concentrations of DNA and MgCl ₂ present in the area above each curve, DN
It shows that A crystallizes.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Toshiyuki Chatake             4 of 2 Shirane, Shikata, Tokai-mura, Naka-gun, Ibaraki Prefecture               Japan Atomic Energy Research Institute Tokai Research Center F-term (reference) 4B024 AA20 CA01 HA11                 4G077 AA01 BF05 CB04 CB06

Claims (9)

[Claims] 1. DNA concentration (mM) and MgCl ₂ located in the region above each curve shown in FIG. 1 in the presence of 20-30% 2-methyl-2,4-pentanediol (MPD). A method for growing a large single crystal of a 10-mer DNA oligomer, which comprises depositing a single crystal of a 10-mer DNA oligomer by a batch method under conditions of concentration (mM). 2. When a graph is prepared in the presence of 30% MPD at a DNA concentration of 5.0 mM or less, with MgCl ₂ concentration (mM) as the X-axis and DNA concentration (mM) as the Y-axis. , A combination consisting of the following (MgCl ₂ concentration, DNA concentration);
(7.5, 5.0); (7.5, 4.5); (15, 1.8); (50, 0.
9); (200, 0.9); (500, 1.8); (600, 3.0); and (600, 5.0); The 10-mer DNA oligomer large size according to claim 1, wherein a single crystal of the DNA oligomer is precipitated by a batch method at a DNA concentration (mM) and an MgCl ₂ concentration (mM) contained in a large region. Single crystal growth method. 3. When a graph is prepared in the presence of 25% MPD at a DNA concentration of 5.0 mM or less, with MgCl ₂ concentration (mM) as the X axis and DNA concentration (mM) as the Y axis. , A combination consisting of the following (MgCl ₂ concentration, DNA concentration);
(50, 5.0); (50, 2.5); (200, 1.0); (300, 1.
5); (600, 4.5); and (600, 5.0); the DNA concentration (mM) contained in the region where Y is larger than the polygonal line formed by connecting each point with a straight line in this order, and (600, 5.0);
The method for growing a large single crystal of a 10-mer DNA oligomer according to claim 1, wherein a single crystal of the DNA oligomer is deposited by a batch method at a MgCl ₂ concentration (mM). 4. When a graph is prepared in the presence of 22.5% MPD and at a DNA concentration of 5.0 mM or less with MgCl ₂ concentration (mM) as the X axis and DNA concentration (mM) as the Y axis. , A combination consisting of the following (MgCl ₂ concentration, DNA concentration);
(100, 5.0); (100, 4.5); (200, 3.0); (400,
3.0); and (400, 5.0); the DNA concentration (mM) and MgCl ₂ concentration (mM) contained in the region where Y is greater than the polygonal line formed by connecting the points in this order with a straight line. 10. The method according to claim 1, wherein the single crystal of the DNA oligomer is deposited by a batch method in the method.
Method for growing large single crystal of oligomeric DNA oligomer. 5. When a graph is prepared in the presence of 20% MPD at a DNA concentration of 5.0 mM or less, with MgCl ₂ concentration (mM) as the X axis and DNA concentration (mM) as the Y axis. , A combination consisting of the following (MgCl ₂ concentration, DNA concentration);
(200, 5.0); (200, 4.5); (400, 4.5); and (400, 5.0); a region in which Y is larger than a polygonal line formed by connecting each point with a straight line in this order. In the DNA concentration (mM) and MgCl ₂ concentration (mM) contained in
The method for growing a large single crystal of a 10-mer DNA oligomer according to claim 1, wherein a single crystal of the DNA oligomer is deposited by a batch method. 6. A single crystal of a DNA oligomer is deposited by a batch method under the conditions of 30% MPD, a 10-mer DNA oligomer having a concentration of 1.5 mM, and MgCl _{2 having} a concentration of 100 mM. The method for growing a large single crystal of a 10-mer DNA oligomer according to claim 1 or 2. 7. The 10-mer DNA oligomer is CCATTAATGG (SEQ
The method for growing a large single crystal oligomer of DNA oligomer according to any one of claims 1 to 6, which has a sequence of ID NO: 4). 8. A large single crystal of a 10-mer DNA oligomer produced by the method according to claim 1 and having physicochemical properties suitable for structural and crystallographic analysis. . 9. Produced by the method of claim 7,
A large single crystal of a DNA oligomer having a sequence of CCATTAATGG (SEQ ID NO: 4), which has physicochemical properties suitable for structural and crystallographic analysis.