JPH07224086A - Method for spreading nucleic acid cluster - Google Patents

Abstract

PURPOSE:To planarly spread a nucleic acid cluster useful as e.g a footing for making fine structures controllable in high accuracy, by charging a solution with the nucleic acid cluster followed by application of an electric field and by taking advantage of the electrostatically attractive force between electric field-forming electrodes and the dipoles produced by the electric field. CONSTITUTION:A nucleic acid cluster 7 is charged in a solution and added and fixed to a desired site of a planar substrate 1 made of e.g. Teflon followed by passing electric current into electric field-forming electrodes 2, 4 to apply an electric field to the cluster to develop, in this cluster 7, a dipole with its vector opposite to that of the electric field to produce electrostatically attractive force between this dipole and the electrodes 2, 4, thus attracting both the left and right ends of the cluster 7 to the electrodes 2, 4, respectively, and spreading the cluster horizontally. Subsequently, an alternate current electric field comparable in intensity to the above-mentioned electric field is applied to generate similar electrostatic attractive force, thus attracting the cluster 7 to extend it perpendicularly. These above-mentioned operations are then repeated several time, thereby planarly spreading the nucleic acid cluster 7 to be used as a footing in making fine structures controllable with accuracy as fine as nanometer order.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for developing a nucleic acid complex to be used as a scaffold for producing a controllable fine structure with nanometer accuracy.

[0002]

2. Description of the Related Art Today's progress in semiconductor microfabrication technology includes
There are some remarkable things, and it is possible to process with sub-micron precision, but in order to perform finer processing, extension of the conventional technology is not enough. There is a strong demand for establishment. In addition, recently, great expectations have been placed on electronic devices that utilize the physical properties of the so-called mesoscopic region of tens to millions of atoms, and biotechnology that uses complexes (supramolecules) of biomolecules such as proteins. There is. This kind of technology also
It is called nanometer technology because it targets extremely small things of the order of nanometers, and it is absolutely necessary to establish a manufacturing technology for fine structures with nanometer precision.

As described above, the need for a technique for producing a fine structure of nanometer accuracy is very high under the present circumstances, and some approaches have already been taken. Good examples are the production of monoatomic thin films by epitaxial growth and the production of monomolecular films by the Langmuir-Blodgett method. Although these methods can control the film thickness with nanometer accuracy, it is difficult to control the orientation and structure in the plane of the film. In addition, there is a scanning tunneling microscope that can be controlled in a plane. If this microscope is used, processing and operation at the level of one atom can be performed, but parallel processing is difficult, making it unsuitable for mass production, and the energy cost is high because a much larger probe must be moved compared to an atom. There is a problem of becoming.

On the other hand, nucleic acid is a linear molecule in which nucleic acid bases are one-dimensionally arranged at a pitch of 0.34 nm. Nowadays, due to advances in artificial synthesis technology and genetic engineering, it is possible to form a nucleic acid having an arbitrary base sequence, and thus it is possible to control the structure of one base unit (0.34 nm unit) at the minimum. Nucleic acid is suitable for mass production because a large number of molecules of picomole (10 ¹¹ ) can be produced at one time, and since it reacts at room temperature, energy cost is low.

However, in order to produce a fine structure using a nucleic acid, it is necessary to use a nucleic acid complex having a two-dimensional spread as a scaffold. 32
The invention of No. 4452 was proposed. The present invention provides multiple types of nucleic acid units each having cohesive ends each having a unique base sequence at a predetermined site, and selectively connecting these nucleic acid units by a hybridization reaction. Although the nucleic acid complex is synthesized by the method described above, the synthesized nucleic acid complex is irregularly rounded or folded, so it is not possible to use it as a scaffold when creating fine structures. It is necessary to spread the nucleic acid complex in a planar manner by the method of.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to propose a practical method for planarly developing a nucleic acid complex used as a scaffold for producing a fine structure.

[0007]

[Means for Solving the Problems] When a synthesized nucleic acid complex is loaded into a solution (for example, a buffer solution) and an AC electric field or a DC electric field is applied to the solution, a dipole is generated inside the nucleic acid by the action of the electric field. An electrostatic attraction force is generated between the dipole and the electric field forming electrode. On the other hand, when the synthesized nucleic acid complex is loaded into a solution and the solution is caused to flow by a pump or other mechanical means, a fluid force acts on the nucleic acid complex. The same applies when the solution loaded with the nucleic acid complex is heated or cooled to cause convective flow in the solution.
The present inventor has conducted an experiment of deploying a nucleic acid complex in a planar shape by utilizing such electrostatic attraction force or fluid force,
It was confirmed that the intended purpose could be achieved sufficiently.

Further, the present inventor conducted an experiment in which fine particles such as polystyrene and metal were bound to desired sites of the synthesized nucleic acid complex and the fine particles were moved using optical tweezers, a scanning tunneling microscope or a similar means. As a result, it was also confirmed that the traction force associated with the movement of the microparticles effectively acts on the nucleic acid complex so that the complex can be developed in a planar shape.

In either case, it is desirable that the nucleic acid complex is developed in at least two directions different from each other. In this way, the nucleic acid complex can be smoothly developed in a planar shape. Further, it is desirable that the nucleic acid complex is subjected to the spreading operation with at least a part thereof fixed to the substrate. In this case, since the nucleic acid complex does not move, the spreading effect is increased, and the nucleic acid complex is There is an advantage that the position of the body does not change.

[0010]

EXAMPLES Hereinafter, the method for developing a nucleic acid complex according to the present invention will be described in more detail with reference to the examples for developing a complex composed of DNA (deoxyribonucleic acid).

<Embodiment 1> In FIG. 1 and FIG.
Is, for example, D synthesized by the method according to the above patent application.
FIG. 3 shows the NA complex, and one corner of the complex is
For example, it is fixed to a desired portion 6 of the flat substrate 1 made of Teflon. The solid lines in FIG. 2 represent individual single-stranded DNAs constituting the nucleic acid complex 7, and the broken lines represent complementary base pairs composed of adenine and thymine or guanine and cytosine.

For fixing the DNA complex 7 to the flat substrate 1, for example, the "Journal of American Chemical Society" is used.
y) "114 (1992) 2656-266
This was done by utilizing the method described on page 3. That is, nucleotides are added to the substrate 1 one by one to produce short DNA fragments bound to the substrate. D
The base sequence of the NA fragment is constructed so as to have a complementary relationship with the base sequence of the single-stranded portion provided at one corner of the DNA complex 7. Therefore, a part of the DNA complex 7 is allowed to undergo a hybridization reaction with a DNA fragment on the substrate 1 so that a part of the complex is formed on the flat substrate 1.
Can be fixed to. However, as it is, the substrate 1
The DNA complex 7 fixed to the above has an irregular shape such as curled up or folded.

Therefore, as shown in FIG. 1, the electrodes 2 to 5 are arranged around the composite 7, and first, an alternating electric field of about 1 MV / m and 1 MHz is applied between the electrodes 2 and 4. As a result, when a dipole opposite to the electric field is generated inside the DNA, an electrostatic attractive force is generated between the dipole and the electrodes 2 and 4, and as a result, the composite 7 has electrodes on both left and right ends. 2 and the electrode 4 are attracted and extend in the horizontal direction in the drawing. Next, when a similar electrostatic attraction force is generated by applying a similar AC electric field between the electrodes 3 and 5, the composite 7 is attracted to the electrodes 3 and 5 by its upper and lower ends. And extend in the direction perpendicular to the drawing. By repeating the same operation a predetermined number of times, the composite body 7 can be developed in a planar shape.

The developed DNA complex 7 is naturally dried or heat dried and fixed on the flat substrate 1, and then, although not shown in detail, a functional substance (protein having a catalytic action or conductivity) is present at a desired site. A fine structure can be produced by adding an engineeringly useful substance such as a gold colloid having a). It is also possible to add the functional substance before the expansion of the complex 7.

The applied electric field may be a DC electric field. However, when a DC electric field is applied, bubbles due to electrolysis are generated on the surface of the electrode and the electric field may be disturbed, which may hinder the development of the DNA complex. Therefore, the AC electric field is usually preferable. In addition, when a DC electric field is applied, since DNA has a net negative charge and is attracted to the positive electrode, it is preferable to apply an AC electric field because the position of the entire DNA complex does not change significantly.

Example 2 This example is different from Example 1 in that the solution is forcibly flowed by using a pump or the like and the DNA complex is developed by utilizing the fluid force. That is, as shown in FIG. 3, after fixing one corner of the DNA complex 7 on the flat substrate 1 by the same procedure as in the case of Example 1, the flow path 14 is formed.
-17 and valves 10-13 are provided. First, the valve 10 and the valve 12 are opened to cause the solution to flow from the flow path 14 toward the flow path 16, and the complex 7 is extended in the horizontal direction in the drawing by utilizing the fluid force. The solution is in the opposite direction (channel 16
→ It is preferable to alternately flow into the flow path 14) because the overall position of the composite 7 does not change significantly. Next, the valve 11 and the valve 13 are opened to cause the solution to flow from the flow path 15 toward the flow path 17, and the composite 7 is also elongated in the vertical direction in the drawing by using the same fluid force. Also in this case, it is preferable that the solution alternately flow in the opposite direction (flow path 17 → flow path 15). By repeating such an operation a proper number of times, D
The NA complex 7 can be developed in a plane.

<Embodiment 3> This embodiment is different from Embodiment 2 in that convection generated by heating or cooling a solution is used. That is, in the present embodiment, as shown in FIGS. 4 and 5, the flat substrate 1 having one corner of the DNA complex 7 fixed at a desired site 6 is loaded inside the container 26 and the periphery of the complex 7 is loaded. The heaters 20 to 23 are installed in the. First, the heater 20 is heated to cause the convection 28 shown in FIG. 5 to occur in the solution 27, and the complex 7 is extended to the left in the drawing by utilizing the fluid force associated with the convection. In this case, it is preferable that the heater 20 and the heater 22 are switched to alternately generate convection in the opposite direction, and the extension on the left side of the drawing and the extension on the right side of the drawing are repeated so that the overall position of the composite 7 does not significantly change. next,
By appropriately using the heater 21 and the heater 23 in combination, convection above and below (see FIG. 4) in the drawing is caused alternately, and the fluid force of the convection is used to move the composite 7 in the vertical direction in FIG. Extend. The DNA complex 7 can be developed by repeating the same operation a suitable number of times.

As means for causing convection, in addition to the above heater, it is possible to use heating means by electromagnetic waves such as light and microwaves, and instead of heating the solution, cooling means such as Peltier element. It is also possible to cool a part of the solution to cause convection. Furthermore,
For example, if a heater and a Peltier element are used together and heating and cooling are combined, the convection of the solution can be made stronger,
The effect of expanding the DNA complex can be further enhanced.

<Embodiment 4> In this embodiment, as shown in FIG. 6, fine particles 29 and 30 of polystyrene having a diameter of about 50 nm to 50 μm are provided at two corners of the DNA complex 7 (bonding portion with the substrate 1). (Excluding) is used. The fine particles 29 and 30 can be connected by the same method as that for fixing the composite 7 to the substrate 1.

First, using optical tweezers composed of a light beam such as a finely focused laser beam, the fine particles 29 are grasped by the tweezers and moved to the right in the drawing, and the traction force generated by the movement of the fine particles is used. The composite 7 is extended in the horizontal direction in the drawing. Next, the fine particles 30 are grasped by the optical tweezers and moved downward in the drawing, and the traction force generated along with the movement of the fine particles is used to extend the composite 7 in the direction perpendicular to the drawing. By alternately repeating these two operations a predetermined number of times or performing them simultaneously, the DNA complex 7 can be developed in a planar shape.

The fine particles are fixed on all three corners of the composite 7 except the bonding portion with the substrate 1, or the composite 7 is not fixed on the substrate 1 and the four corners of the composite are removed. If the fine particles are fixed to the whole and these fine particles are moved in the radial direction from the center of the complex 7, the expansion effect can be further increased. It is also possible to use metal particles such as gold instead of polystyrene particles, and to use a scanning tunneling microscope or a similar device (scanning microprobe microscope) instead of optical tweezers. While the position control accuracy of the optical tweezers is several hundred nm, which is about the same as the wavelength of light, the position control accuracy of the scanning tunneling microscope is about 0.1 nm, which is about several thousand times higher, enabling finer movement operations. Is.

<Supplementary Explanation> In the above-mentioned embodiment, the case where the DNA complex is developed in two directions orthogonal to each other has been explained. However, in general, the DNA complex is developed in at least two directions different in direction. Good. In the case of a DNA complex that is particularly long in one direction, a sufficient effect can be expected even if it is developed only in the long axis direction.

In the above embodiment, the case where one corner of the DNA complex is fixed on the substrate has been described.
The DNA complex does not necessarily have to be immobilized on the substrate. However, when immobilized on a flat substrate, it is possible to prevent the movement of the entire DNA complex, which is advantageous in that the expansion effect is great and the position of the DNA complex does not change.

Further, in the above-mentioned embodiment, the case of using the DNA complex was explained, but the developing method of the present invention is
Not only the complex consisting of NA (ribonucleic acid), but also DNA
In addition to RNA, DNA or RN chemically modified
The same can be applied to a general nucleic acid complex containing A.

[0025]

EFFECTS OF THE INVENTION According to the present invention, it is possible to develop a nucleic acid complex having an irregular shape such as curling or folding and shaping it into a planar shape. Moreover, the nucleic acid complex developed in a plane according to the present invention can be effectively used as a scaffold for producing various planar fine structures controlled with nanometer accuracy.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a first embodiment of the present invention.

FIG. 2 is a plan view showing a DNA complex immobilized on a flat substrate.

FIG. 3 is a schematic diagram showing a second embodiment of the present invention.

FIG. 4 is a schematic diagram showing a third embodiment of the present invention.

FIG. 5 is a sectional view for explaining a third embodiment of the present invention.

FIG. 6 is a schematic diagram showing a fourth embodiment of the present invention.

[Explanation of symbols]

1 ... Planar substrate, 2-5 ... Electrode, 6 ... Substrate fixing part, 7 ...
DNA complex, 10 to 13 ... Valve, 14 to 17 ... Flow path, 20 to 23 ... Heater, 26 ... Container, 27 ... Solution,
28 ... convection, 29, 30 ... fine particles.

Claims (10)

[Claims] 1. A nucleic acid complex is loaded into a solution, an electric field is applied thereto, and the electrostatic attraction force generated between the dipole generated inside the nucleic acid complex by the electric field and the electric field forming electrode is utilized. A method for developing a nucleic acid complex, which comprises developing the nucleic acid complex. 2. The method for developing a nucleic acid complex according to claim 1, wherein the applied electric field is an alternating electric field. 3. The method for developing a nucleic acid complex according to claim 1, wherein the applied electric field is a direct current electric field. 4. A method for developing a nucleic acid complex, which comprises loading the nucleic acid complex into a solution and developing the nucleic acid complex by utilizing a fluid force generated by flowing the solution. 5. The method for developing a nucleic acid complex according to claim 4, wherein the flow of the solution is convection. 6. A nucleic acid which is characterized in that fine particles are fixed at a desired site of a nucleic acid complex and loaded into a solution, and the traction force generated by moving the fine particles is used to develop the nucleic acid complex. How to deploy the complex. 7. The method for developing a nucleic acid complex according to claim 6, wherein the moving means of the fine particles is optical tweezers. 8. The method for developing a nucleic acid complex according to claim 7, wherein the moving means of the fine particles is a scanning tunneling microscope or a device similar thereto. 9. The method of developing a nucleic acid complex according to claim 1, wherein the nucleic acid complex is developed in at least two different directions. 10. The development of the nucleic acid complex according to any one of claims 1 to 9, wherein the nucleic acid complex is developed with at least a part of the nucleic acid complex fixed to a substrate. Method.