DNA paste, DNA plastic and manufacturing method thereof, DNA thin film and formation method thereof, and DNA thin film pattern and formation method thereof

Abstract

To solve problems such as the deterioration of the marine environment, including marine ecosystems, due to marine litter in recent years, particularly the problem of marine plastic litter, which not only occurs in large quantities every day around the world but also remains in the ocean for long periods of time and requires global-scale measures, and problems such as the reuse of industrial waste, such as salmon milt, which amounts to approximately 10,000 tons per year and is disposed of as industrial waste in Japan, in order to realize a sustainable society.SOLUTION: With the aim of potentially reusing the DNA contained in salmon milt and reducing the burden on the marine environment in mind, we focused on DNA not as a gene but as a polymeric material, leading to the invention of a DNA paste that could be used in enzymatically degradable plastics and thin film applications in nanoelectronics. Specifically, dialdehyde is added to an aqueous solution of DNA salt, and the paste is then baked at a temperature above the melting point of DNA, resulting in crosslinking.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a DNA paste and a method for synthesizing the same, which is characterized by adding dialdehyde to an aqueous solution of a salt of deoxyribonucleic acid (hereinafter referred to as DNA) derived from marine organisms such as salmon milt, and baking the solution at a temperature above the melting temperature (hereinafter referred to as Tm) of DNA; an enzymatically degradable DNA plastic obtained by curing the DNA paste (defined below) and a method for producing the same; an enzymatically degradable DNA thin film obtained by film formation and curing using the DNA paste (defined below) and a method for forming the same; and an enzymatically degradable DNA thin film pattern obtained by exposing the DNA thin film to ultraviolet light (hereinafter referred to as UV) through a photomask and then washing with water to selectively remove only the UV-exposed areas, and a method for forming the same. [Background technology]

[0002] In recent years, marine litter has been causing problems such as the deterioration of the marine environment, including marine ecosystems. In particular, marine plastic litter is not only being generated in large quantities every day around the world, but also remains in the ocean for long periods of time, making global-scale measures necessary.

[0003] In Japan, more than 100,000 tons of salmon are caught annually, and while the fish meat, often sold as fillets, and salmon roe (ikura) are eaten, the sperm (milt) is disposed of as industrial waste, amounting to approximately 10,000 tons per year. Reusing this industrial waste is a challenge in realizing a sustainable society. Examples of applications for DNA isolated and extracted from discarded salmon milt include medical materials such as medicines and nutritional supplements, but the market for this is small, and most DNA is treated as industrial waste.

[0004] We focused on the possibility of reusing the DNA contained in salmon milt, and attempted to develop it not as a gene but as a polymer material. We also considered applying it to enzymatically degradable plastics and functional thin films to reduce the burden on the marine environment. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2003-073925 [Non-patent literature]

[0006] [Non-Patent Document 1] Research on optical materials from DNA derived from marine organisms, Naoya Ogata, Polymer Journal, Vol.61, No.1, pp.22-28 (Jan., 2004) [Non-patent document 2] EL devices made from salmon-derived DNA, Yoshio Okahata, Takemi Kawasaki, Polymers, Vol. 55, July issue (2006)

[0007] When considering the reuse of DNA derived from marine organisms such as salmon milt in plastics or functional thin films, the water solubility of DNA poses a problem. This is because DNA dissolves when exposed to moisture in the air or when wet with rain. To overcome this drawback, many studies have focused on the negative charge of the phosphate groups facing outward in the double helix structure as the reactive site of DNA. Numerous reports have been published on a method (hereafter referred to as the casting method) in which an aqueous solution of sodium salt of DNA (hereafter referred to as DNA-Na) is reacted with cationic lipids, or the hydrophobic DNA-lipid complex obtained by reacting the solution with the cationic lipids is dissolved in a solvent, spread on a substrate, and the solvent is evaporated to obtain a thin film.

[0008] [Non-patent document 3] Effect of Lipids on the Physical Properties of DNA-Lipid Complexes, Hiroshi Yamaoka, Naoya Ogata, Journal of Polymer Science, Vol. 61, No. 7, pp. 384-390 (Jul., 2004)

[0009] While lipids are lipophilic (soluble in organic solvents), which avoids the problem of DNA solubility, the use of cationic lipids and large amounts of organic solvents is environmentally unfriendly. Furthermore, thin films prepared by the casting method generally have the disadvantages of being difficult to scale up and of being less uniform in thickness than thin films prepared by methods such as spin coating. Summary of the Invention [Problem to be solved by the invention]

[0010] The first object of the present invention is to provide a DNA paste (defined below) that can be enzymatically decomposed and can be used to form thin films using high-speed rotation (centrifugal force) such as spin coating, and a method for synthesizing the same, which is prepared by adding dialdehyde to an aqueous solution of DNA salt in water and baking the resulting solution at a temperature above the melting temperature of DNA.

[0011] Enzyme-degradable DNA plastics made using the DNA paste can be developed as an alternative plastic with reduced environmental impact, as they can prevent the deterioration of the marine environment caused by marine plastic waste. Furthermore, by forming thin films of the enzymatically degradable DNA plastics, they can be used as photosensitive materials for photoresists in the semiconductor field, reducing the environmental impact. Therefore, a second object of the present invention is to provide an enzymatically degradable DNA plastic obtained by curing the DNA paste (defined below) and a method for producing the same, an enzymatically degradable thin film using the DNA paste and a method for forming the same, and further to provide a pattern of the enzymatically degradable DNA thin film obtained by exposing the DNA thin film to UV light and developing it with water. [Means for solving the problem]

[0012] When considering the development of thin films for DNA reuse, chemical reactions with DNA are also important. The present invention provides a DNA paste (defined below) that is enzymatically degradable and can be formed into thin films by spin coating, without using environmentally hazardous cationic lipids, by crosslinking DNA molecules to make them insoluble in water. It also provides a method for synthesizing the DNA paste. The present invention also provides an enzymatically degradable DNA plastic obtained by curing the DNA paste (defined below) and a method for producing the same. Furthermore, for application in the semiconductor field, the present invention also provides an enzymatically degradable DNA thin film obtained by film formation and curing using the DNA paste, and a method for forming the same. This could potentially be used as a photosensitive material for photoresists with reduced environmental impact. The present invention focuses on the fact that DNA has a UV absorption maximum wavelength near 260 nm. Specifically, exposure to UV light near 260 nm breaks the crosslinks between DNA molecules, returning them to water-soluble DNA, enabling development using water. This development process also eliminates the need for environmentally hazardous developing solutions, further enhancing environmental benefits. During UV exposure, a photomask with a desired pattern printed in black is inserted between the UV irradiation device and the substrate formed with DNA paste (defined below), making it possible to obtain a DNA thin film with the desired pattern. This provides a pattern of an enzymatically degradable DNA thin film and a method for forming it.

[0013] In this study, we focused on the amino groups of bases facing inward in the double helix structure as reactive sites of DNA and came up with the idea that crosslinks between DNA strands could be formed by reacting them with dialdehyde. However, because bases typically face inward in the double helix structure, simply adding dialdehyde does not result in a reaction. We then discovered that by raising the temperature of the DNA solution above its Tm, more than half of the DNA dissolves from its double-stranded structure to a single-stranded structure, making it possible to react with the aldehyde groups. Specifically, adding dialdehyde to a DNA-Na solution and baking it at a temperature above Tm for a predetermined time results in the formation of crosslinks between DNA strands (i.e., imine or enamine formation), resulting in a paste-like viscous liquid. In this study, we define the "paste-like viscous liquid obtained by adding dialdehyde to a DNA-Na solution and baking it at a temperature above Tm for a predetermined time to form crosslinks between DNA strands" as "DNA paste." To experimentally determine the Tm of DNA with a given number of base pairs, it is common to measure absorbance using a UV-visible spectrometer with temperature as a variable and analyze the profile, which requires an excessive amount of time. In contrast, the Tm can be calculated easily and accurately using the GC% method, so the present invention employs the Tm based on this method.

[0014] [Non-patent document 4] Basic Lectures <The Fusion and Interface of Bio and Electronics> Fundamentals of Genetic Engineering I -From the Basics of DNA Manipulation to Genetic Recombination Experiments-, Takaaki Tamura, Applied Physics, Vol. 74, No. 1, pp. 384-390 (2005)

[0015] For example, commercially available DNA-Na derived from common salmon milt has approximately 1000 base pairs and a GC% of 41.2%. By applying these values ​​to the GC% method, the Tm of the DNA in aqueous solution can be calculated as 72.1°C. Dialdehydes include glyoxal (boiling point: 50.4°C), malondialdehyde (boiling point: 74°C), succinaldehyde, glutaraldehyde (boiling point: 188°C), and adipaldehyde (boiling point: 153.66°C). However, those with boiling points lower than the Tm of DNA are unsuitable. Furthermore, taking into account volatility, glutaraldehyde is clearly the preferred crosslinker for commercially available DNA-Na derived from common salmon milt. Needless to say, the viscosity of the resulting DNA paste can be adjusted by controlling the baking temperature and time.

[0016] The DNA paste is dropped onto a substrate of your choice, coated onto the substrate by spin coating, and cured at a predetermined temperature, allowing for the formation of a thin DNA film. The thickness of the thin DNA film can be controlled by the rotation speed of the spinner during spin coating. When applying photolithography to the thin DNA film, the thin DNA film can be exposed to UV light with a wavelength of approximately 260 nm through a black photomask bearing the desired pattern. Then, development using water allows for the formation of a thin DNA film with the desired pattern. [Effects of the Invention]

[0017] In the present invention, a DNA paste with reduced environmental impact is obtained by using a crosslinking reaction between the amino group of the base facing inward in the double helix structure and an aldehyde group as the reactive site of DNA. Furthermore, this DNA paste enables the application of spin coating, which not only allows for the large-area deposition of DNA thin films but also significantly improves the uniformity of the film thickness. While spin coating is used here as an example of a film formation method, it goes without saying that this method is not limited to this method. Furthermore, by including a step of exposing this DNA thin film to UV light with a wavelength of approximately 260 nm through a photomask with the desired pattern printed on it and developing it with water, it is possible to obtain a DNA thin film with the desired pattern, which is expected to be applied to photolithography technology in the nanoelectronics field. [Brief explanation of the drawings]

[0018] [Figure 1] 1 is a photograph showing the appearance of the DNA paste prepared by the synthesis method of Example 1. [Figure 2] 1 is a photograph showing the appearance of a 3.5 μm-thick DNA thin film formed on a slide glass by the method of Example 3. [Figure 3] This figure shows the time dependence of enzymatic degradation of a thin DNA film formed by the method of Example 3. The horizontal axis represents the elapsed time, and the vertical axis represents the amount of enzymatic degradation (expressed as %) when the maximum absorbance at a wavelength of 234 nm is taken as 100%. Squares represent a micrococcal nuclease concentration of 40 U / mL, and circles represent a micrococcal nuclease concentration of 0 U / mL (no micrococcal nuclease) for comparison. [Figure 4] 1 shows the exposure time dependence of the film thickness and film weight of the DNA thin film formed by the method of Example 3, where the horizontal axis represents the exposure time and the vertical axis represents the film thickness and film weight. DETAILED DESCRIPTION OF THE INVENTION

[0019] Examples are shown below, but it goes without saying that the present invention is not limited thereto. Example 1 shows an example of "DNA paste and a synthesis method thereof," Example 2 shows an example of "enzyme-degradable DNA plastic and a method for producing it," Example 3 shows an example of "enzyme-degradable DNA thin film and a method for forming the film," and Example 4 shows an example of "enzyme-degradable DNA thin film pattern and a method for forming the pattern." [Example]

[0020] Commercially available salmon milt-derived DNA-Na has a base pair count of approximately 1000 and a GC% of approximately 41.2%. First, 50 mg of this material was weighed out using a precision balance and placed in a vial. 5 mL of 25% glutaraldehyde solution (as a dialdehyde) was added dropwise, and the vial was left overnight with the lid closed. The vial was then heated for 5 hours at 80°C, a temperature higher than the Tm (72.1°C; calculated by the GC% method) of the DNA solution. After heating, the DNA was carefully dropped near the center of a PTFE sheet using a Pasteur pipette. The sheet was then placed in a Petri dish, covered, and left overnight to synthesize the low-viscosity DNA paste shown in Figure 1. While the heating conditions used here were 80°C for 5 hours, the heating conditions may be adjusted depending on the desired viscosity of the DNA paste. [Example]

[0021] Approximately 6 to 10 drops of the DNA paste synthesized in Example 1 were carefully dispensed near the center of a PTFE sheet using a Pasteur pipette. This was placed in a Petri dish, uncovered, and cured at 70°C for 30 minutes in an inert oven with nitrogen gas introduced into the chamber, producing DNA plastic. The enzymatic decomposition of DNA plastic produced using this method is described in Example 3. [Example]

[0022] First, a glass slide was used as the substrate. It was ultrasonically cleaned with ethanol, rinsed with water, dried, and then placed in the center of the spin coater's substrate support. Here, a glass slide was used as the substrate, but any desired substrate may be used. Next, approximately 6–10 drops of the DNA paste synthesized in Example 1 were carefully dispensed onto the center of the substrate using a Pasteur pipette. The film formation conditions for the spin coater were a maximum rotation speed of 2000 rpm and 50 seconds. After spin coating, the substrate was placed in a Petri dish, uncovered, and cured at 70°C for 30 minutes in an inert oven with nitrogen gas introduced into the chamber, resulting in the formation of a uniform DNA thin film with a thickness of 3–15 μm. The thickness of the DNA thin film can be controlled by the viscosity of the DNA paste synthesized in Example 1. For example, Figure 2 shows an example of a photograph of the appearance of a 3.5 μm-thick DNA thin film formed.

[0023] Here, the DNA thin film formed on a glass slide using the aforementioned film formation method was cut into 5 mm squares using a diamond cutter and placed in a vial. Then, 10 mL of pH 8.0 phosphate buffer was added. 20 μL of the DNA-degrading enzyme micrococcal nuclease was added dropwise using a micropipette to achieve a concentration of 40 U / mL. The vial was then capped and placed in an oven at 37°C. After a predetermined time, the supernatant was sampled using a Pasteur pipette, and its absorbance was measured using a UV-visible spectrophotometer. The detection wavelength was 234 nm, the maximum absorption wavelength of 3'-mononucleotides produced by micrococcal nuclease degradation.

[0024] The behavior of the squares in Figure 3 shows that the amount of enzymatic decomposition increases with measurement time, reaching a nearly constant value of 100% after about 120 hours. At this point, the disappearance of the DNA thin film can be visually confirmed, demonstrating that the DNA thin film formed using the DNA paste of the present invention can be enzymatically decomposed, and that the DNA plastic of Example 2, which was prepared under the same conditions except for the application of the spin coating method, can also be enzymatically decomposed. [Example]

[0025] The DNA thin film formed on the slide glass in Example 3 was cut into 1 cm squares using a diamond cutter, and the surface of the DNA thin film was irradiated with a UV-LED at a wavelength of 280 nm and an irradiation intensity of 100 mW / cm. 2 The film was irradiated with UV light adjusted to 100% for a specified period of time (hereinafter referred to as exposure). Afterwards, it was washed with water (corresponding to development) and dried, and the film thickness and weight were measured and the appearance was observed under a microscope. The UV wavelength used for exposure here is different from 260 nm, the maximum absorption wavelength of DNA, because the shortest wavelength of UV-LEDs currently available on the market is 280 nm. It goes without saying that in the future, as UV-LED wavelengths are shortened and 260 nm products become more common, exposure times will be shortened by using them.

[0026] As shown in Figure 4, the DNA thin film formed on the slide glass decreased with exposure time and completely disappeared after approximately 120 hours. Furthermore, the disappearance of the DNA thin film was also visible under a microscope, confirming that a DNA thin film formed using the DNA paste of the present invention as a photoresist can be developed by exposing it to ultraviolet light with a wavelength of 280 nm and using pure water as a developer. This clearly demonstrates that patterning of DNA thin films is possible by performing exposure and development through a photomask. [Industrial Applicability]

[0027] It is expected that the added value of being enzymatically degradable can be provided to industries that handle resin and plastic raw materials, as well as industries that process and mold plastics. Furthermore, since water can be used as a developer and the thin film itself is enzymatically degradable, it will enable photolithography with a reduced environmental impact, which is also expected to be applied to the nanoelectronics field.

Claims

1. A DNA paste and a method for synthesizing the same, which are characterized by adding dialdehyde to an aqueous solution of DNA salt in water and baking the solution at a temperature above the melting temperature of DNA.

2. An enzymatically degradable DNA plastic, characterized by using the DNA paste of claim 1, and a method for producing the same.

3. An enzymatically degradable thin DNA film, which comprises the DNA paste of claim 1, and a method for forming the thin film.

4. 4. An enzymatically decomposable DNA thin film pattern and a method for forming the pattern, comprising the steps of exposing the DNA thin film of claim 3 to ultraviolet light through a photomask and subsequently washing it with water.

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