JP2008155211A - Method of producing water insoluble cross-linked dna by ultraviolet irradiation and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a water insoluble cross-linked DNA and a method of using the same as an environment cleaning material. <P>SOLUTION: When ultraviolet rays having a wavelength within the range of 250-270 nm are irradiated on a solution, a solution film, or a thin film of a water soluble DNA (e.g. a DNA originated out of a salmon testis) on a support medium, the DNA is changed into a water insoluble cross-linked body fixed on the support medium. Similarly, for example, when a cellulose non-woven cloth immersed in an aqueous solution of a water soluble DNA is irradiated by ultraviolet rays having a wavelength within the range of 250-270 nm, a cellulose non-woven cloth covered with a water insoluble cross-linked polymer of the DNA is obtained. The cross-linked body adsorbs an intercalating agent into the DNA or an endocrine disrupter at an excellent yield even on the condition that the concentration is very low. Likewise, a toxic metal such as a Hg ion is fixedly adsorbed against water. Therefore, the cross-linked body is useful as an environment cleaning material. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing a water-insoluble crosslinked product of water-soluble DNA fixed to a support by irradiating the water-soluble DNA which may be in an aqueous solution on the support with ultraviolet rays having a wavelength in the range of 250 to 270 nm. The present invention relates to a pharmaceutical, veterinary medicine or food containing the cross-linked product; accumulation and removal of an intercalating agent into DNA by the cross-linked product; and the use of the cross-linked product containing silver ions as a sterilizing material.

  It is known that double-stranded DNA crosslinks between each double-stranded DNA in ethanol by ultraviolet irradiation (see, for example, Photochemistry and Photobiology, 1998, 67 (4): 386-390). . However, it is not known that cross-linking occurs between double-stranded DNA under water or solvent-free conditions. In addition, crosslinking of single-stranded DNA or RNA by ultraviolet irradiation is not known.

  The present inventor has examined the influence of ultraviolet irradiation of water-soluble double-stranded DNA, water-soluble single-stranded DNA or RNA in water or in the absence of a solvent. It was discovered that a water-insoluble cross-linked product between the single-stranded DNA and RNA was produced, and the present invention was completed based on this discovery.

  That is, the present invention is obtained by concentrating or drying a water film of a water-soluble DNA on a support or a liquid film thereof, or a thin layer of water-soluble DNA on a support or a liquid film of a water-soluble DNA solution on a support. The present invention relates to a method for producing a water-insoluble crosslinked polymer of water-soluble DNA fixed to a support by irradiating the obtained thin layer with ultraviolet rays having a wavelength in the range of 250 to 270 nm.

  The present invention also provides a water-insoluble cross-linking of water-soluble DNA by irradiating an aqueous solution of water-soluble DNA immersed in a support, for example, a cellulose fiber nonwoven fabric, with ultraviolet rays having a wavelength in the range of 250 to 270 nm. It also relates to a method for producing a support or cellulose fiber nonwoven fabric coated with a polymer.

The present invention also provides a water-insoluble cross-linked polymer of water-soluble DNA immobilized on the support, and a water-insoluble cross-linked polymer obtained by removing the support from the water-insoluble cross-linked polymer of water-soluble DNA immobilized on the support by, for example, peeling. It also relates to the use of cross-linked polymers.
The present invention is also applicable to a tobacco filter or an air purifier for removing an intercalating agent into double-stranded DNA in the air or tobacco smoke, characterized by containing double-stranded DNA. It also relates to other gas filter media.
The present invention also relates to the gas filter material as described above, wherein the intercalating agent for double-stranded DNA is benzo [a] pyrene, benzo-p-dioxane, dibenzofuran and biphenyl.

  Examples of water-soluble DNA include single-stranded DNA or double-stranded DNA, such as DNA obtained from fish testis or animal thymus. For example, those obtained from salmon, herring, cod roe (testis) are preferred. Further, mammals or birds such as those obtained from thymus such as cows, pigs and chickens are preferred. Examples of other water-soluble DNAs may be synthetic DNAs, particularly DNA sequences having base pairs of (dA)-(dT), particularly DNAs having a poly (dA) -poly (dT) type sequence, for example. . As an example of RNA, RNA obtained from yeast is preferable.

  The support is only required to be capable of supporting the water-insoluble crosslinked product of the produced water-soluble DNA in the form of a film, line or dot. The shape and material of the support may be porous, plate-like, spherical, for example spherical or fibrous with a diameter of 0.1 mm to 10 mm, synthetic resin, glass, ceramics or metal; natural fiber such as cellulose or pulp; Alternatively, natural fibers such as cellulose or pulp are chemically processed.

  As a material of the support, a material having poor binding force with a DNA cross-linked body such as glass, synthetic resin such as polyethylene or polypropylene or metal, or a surface-treated glass having a surface having a strong binding force, surface-treated It may be a synthetic resin that may be present, a metal that may be surface-treated, or cellulose. In any case, those which are difficult to be decomposed by the irradiated ultraviolet rays are preferable. The support having a weak binding force with the water-insoluble crosslinked product may be easily peeled from the produced DNA crosslinked product, for example, the film. The support in this case can also be a physical reinforcing material for the crosslinked body when the crosslinked body is not peeled off. Furthermore, the support having a strong binding force with the water-insoluble crosslinked body can also be a physical reinforcing material for the crosslinked body, for example, a film-like crosslinked body.

Glass and ceramics may be those whose surfaces are chemically treated. For example, in the case of commercially available bolus glass beads not subjected to surface treatment (particle size = about 0.5 to 1.2 mm, average about 1 mm), it is immersed in a DNA solution of 10 mg / mL for 2 hours. In the case of surface treatment, for example, porous glass beads are immersed in a solution of concentrated H ₂ SO ₄ : 30% H ₂ O ₂ = 7: 3 at about 70 ° C. for 30 minutes to introduce hydroxyl groups on the surface, and then 10 mg / Immerse in mL of DNA solution for 2 hours. In the same manner, the porous glass beads were concentrated H ₂ SO ₄ : 30% H ₂ O ₂ = 70 ° C.
Immerse it in a 7: 3 solution for 30 minutes, then leave it in a sealed container with 3-aminopropylnitriletriethoxysilane for about 2 hours to introduce organic amino groups on the surface of the beads, and then add it to a 10 mg / mL DNA solution. Immerse for 2 hours.

  The thin layer of water-soluble DNA on the support is usually produced by concentrating or drying the liquid film of the water-soluble DNA solution on the support. The solvent of the solution used at this time is water, aqueous methanol, or aqueous ethanol, and those that are easily air-dried are preferable. Water is particularly preferable. The thickness of the water-soluble DNA thin layer is usually 0.01 to 1 mm, but may be a thickness outside this range.

  The concentration of the aqueous solution of water-soluble DNA may be not more than the concentration of the saturated solution of the aqueous DNA. For example, the concentration is about 10 mg of DNA / 1 mL of water, which is a concentration that facilitates dissolution treatment. The thickness of the liquid film of the aqueous solution of water-soluble DNA is usually 0.1 to 20 mm, preferably 0.1 to 10 mm, but may be outside this range.

The wavelength of the irradiated ultraviolet light is preferably in the range of 250 to 270 nm. The necessary irradiation time is inversely proportional to the irradiation intensity of the irradiated ultraviolet rays. In the case of 5600 μW / cm ² (irradiation distance is about 7.5 cm), the irradiation time is approximately 12 hours or longer. If the irradiation time is insufficient, the yield of water-insoluble DNA decreases. Therefore, it is preferable to select the irradiation intensity and irradiation time with high yield by tracking the amount of aqueous DNA or water-insoluble DNA in the produced film. For example, a DNA film obtained from salmon testis (a solution of 10 mg of DNA and 20 mL of water was dried. The film area was about 10 cm ² ) with 260 nm ultraviolet light having an intensity of 5600 μW / cm ² (irradiation distance was about 7.5 cm). When irradiated, about 50% of DNA is eluted by water from the film produced after 1 hour of irradiation, but about 25% of DNA is eluted after 12 hours of irradiation. The thickness of the thin film is preferably such that the irradiated ultraviolet rays can reach the deep part of the DNA film. Usually, the thickness of the thin film is about 0.01 to 1 mm. The irradiation temperature of ultraviolet rays is usually preferably 0 to 50 ° C. or less, and preferably room temperature, for example, 10 to 35 ° C.

Since the water-insoluble DNA film obtained after elution of water-soluble DNA from the obtained film with water after ultraviolet irradiation is insoluble in water even when immersed in room temperature water for a long period of, for example, 50 days, It is stable and insoluble in water. Water insoluble is defined as not easily dissolved in water at 80 ° C. or lower. The water-insoluble crosslinked product was dissolved in boiling water at 100 ° C. in 15 minutes. Even when this dissolved product was cooled, precipitation into water could not be observed. This dissolved sample is diluted with water to make a solution with a concentration of 0.1 μg / 10 μL, and then agarose gel electrophoresis (manufactured by Washina Sheng (manufactured), mobile phase TAE buffer, analysis temperature 30 ° C., moving distance 12 cm) Analysis showed that it was a very large DNA compound with a very high molecular weight.

Considering the high molecular weight that cannot be measured as water-insoluble at room temperature (25 ° C.), this water-insoluble DNA is presumed to be a DNA cross-linked polymer produced by a cross-linking reaction between a large number of double-stranded DNAs.

  The obtained water-insoluble cross-linked film (of water-soluble DNA) is the raw material used in IR (KBr tablet method) spectrum and UV-VS region absorption spectrum when the water-soluble DNA is derived from salmon testis. It was not possible to recognize any significant difference from that of the water-soluble DNA. At the present time, it is unclear which part is cross-linked.

Accordingly, electrophoresis using Poly (dA) -Poly (dT) (1% agarose gel was performed. As a result, bands were observed in the vicinity of 2.7, 4.3, 5.0 and 6.0 (kbp). The film was irradiated with UV light at 254 nm for 30 minutes, and a water-insoluble film (analysis by electrophoresis using the above 1% agarose gel did not show a band, probably due to insolubility in water). occured. The IR spectrum of this water-insoluble film (film was measured from 20 μg + KBr several hundred g) showed a difference in absorption from the raw material before ultraviolet irradiation in the following wavelength region.
(1) Absorption of νCH derived from the thymine double bond near 1680 cm ⁻¹ (ν: stretching vibration) disappeared.
(2) A large change was ^observed around 1450 cm- ¹ .
(3) 968 cm ^-1 : disappearance of νC-C in deoxyribose (4) 800 cm ^-1 : change in νP-O (5) 1230,1090
cm ^-1 : non-target νPO ₂ Conclusion: The disappearance of absorption near 1680 cm ^-1 indicates the formation of thymine dimer.

  Considering from the analysis result of the above Poly (dA) -Poly (dT), since the (dA)-(dT) pair is present at random in salmon-derived DNA, there is little chance of cross-linking, and water insolubilization occurs. However, it seems that no significant difference was observed in the IR spectrum. From the data obtained at the present time alone, it cannot be determined that only the formation of thymine dimer is the cause of insolubilization.

  The inventor has obtained a film of a water-insoluble crosslinked product thus obtained from a compound such as an environmental hormone that is an intercalating agent for DNA, such as ethidium bromide (4 ppm); benzo [a] pyrene. (10 ppb); dibenzo-p-dioxane (10 ppb); very high yields of these intercalants from aqueous solutions of very low concentrations of dibenzofuran (10 ppb) and biphenyl (10 ppb) [numbers in parentheses described after each compound] Was found to be adsorbed and removed from these aqueous solutions. This effect makes it possible to remove intercalation agents into very low concentrations of DNA present in in vitro or edible liquids or solids or in liquids or gases in the environment.

Therefore, it is useful for removing the insertion agent by appropriately selecting the material and shape of the support. Filter media or adsorbents for collecting and removing intercalating agents such as tobacco filters, air filter gas filter media, drinking water, edible water, beverage foods such as milk, liquid milk milk filters, animals, especially humans. It is useful as an intercalating agent in the digestive tract of mammals, for example, as an adsorption / cleaning agent.

As described above, the support may be a glass and ceramic whose surface is chemically treated. For example, in the case of commercially available bolus glass beads not subjected to surface treatment (particle size = about 0.5 to 1.2 mm, average about 1 mm), after being immersed in a 10 mg / mL DNA solution for 2 hours, dried, Irradiate with ultraviolet light for 2 hours. During the ultraviolet irradiation, porous glass was stirred every 15 minutes. In the case of the surface treatment, for example, porous glass beads are concentrated H ₂ SO ₄ at about 70 ° C.
: Soaked in a solution of 30% H ₂ O ₂ = 7: 3 for 30 minutes to introduce hydroxyl groups on the surface, immersed in a 10 mg / mL DNA solution for 2 hours, dried, and then irradiated with ultraviolet rays in the same manner as described above. Similarly, the porous glass beads were added to a solution of concentrated H ₂ SO ₄ : 30% H ₂ O ₂ = 7: 3 at about 70 ° C.
Soaked for 0 minutes, then left in a sealed container with 3-aminopropylnitrile triethoxysilane for about 2 hours to introduce organic amino groups on the surface of the beads, then immersed in a 10 mg / mL DNA solution for 2 hours and dried In the same manner as described above, ultraviolet irradiation is performed. The amount of DNA immobilized on the surface of these porous glass beads was as follows. Commercially available porous glass beads: 4-6 mg / g of beads; Porous glass beads with hydroxyl groups on the surface: about 3 mg / g of beads; Porous glass beads with organic amino groups on the surface: 5-7 mg / g of beads. The DNA cross-linked product thus immobilized has the same action as the above water-insoluble cross-linked film on the DNA intercalating agent.

The production method of the present invention was applied to a cellulose fiber nonwoven fabric immersed in an aqueous solution containing aqueous DNA. That is, it was found that the water-insoluble crosslinked DNA was immobilized on cellulose as a result of irradiating the immersion liquid with ultraviolet rays in the range of 250 to 270 nm. This cellulose fiber nonwoven fabric functions as a physical reinforcing agent for the water-insoluble crosslinked product. This can adsorb the above intercalating agent or environmental hormone type compound. Moreover, the cellulose fiber nonwoven fabric adsorbed the water-soluble silver salt such as silver sulfate and silver nitrate, for example, from an aqueous solution of silver nitrate so that Ag ⁺ ions could not be washed and removed with water. Then, the free Ag ⁺ non-woven fabric has showed significant growth inhibitory effect on bacteria such as Escherichia coli and Staphylococcus aureus. This result indicates the possibility that metal ions such as Hg ⁺ , Hg ²⁺ , Cu ⁺ and Cu ²⁺ can be used in the same manner in addition to biocidal metal ions such as Ag ⁺ .

  Instead of the above cellulose fiber nonwoven fabric, a normal cellulose fiber fabric, for example, a gauze-like one can also be used. By adjusting the spacing between the cellulose fibers, the size of the nonwoven fabric or woven fabric can be freely adjusted for liquid passing or aeration. Similarly, the cellulose fiber nonwoven fabric can be replaced with cellulose paper. In this case, cellulose paper having a desired pore size may be used. The cellulose may be a cellulose derivative such as methyl cellulose, ethyl cellulose, 2-hydroxyethyl cellulose, or hydroxypropyl cellulose.

Further, the water-insoluble cross-linked product of the water-soluble DNA is a cross-linked product of DNA that maintains the DNA structure, and thus has a binding force to the same metal as DNA. Therefore, it also has a function as an environmental purification material capable of adsorbing metal ions such as Cd ²⁺ , chromium ions such as Cr ³⁺ , Hg ²⁺ , manganese ions such as Mn ²⁺ . This is also true for the water-insoluble crosslinked film of water-soluble DNA and a support coated with the crosslinked body, such as a cellulose fiber nonwoven fabric.

In addition, the water-insoluble crosslinked product of the present invention has been proved not to be hydrolyzed by a certain kind of nuclease (micro-coccal nuclease (Sigma)) in an initial test. This suggests that this is different from ordinary DNA and is not degraded in the digestive tract of mammals and birds including humans. Therefore, the above intercalating agent or metal ions in the digestive tract are accumulated and collected.
It functions as a medicine or veterinary medicine or food having the action of removing poisons, that is, detoxification.

  It has been confirmed that even when the water-soluble double-stranded DNA is replaced with water-soluble single-stranded DNA or RNA, a water-insoluble crosslinked product is formed as a result of irradiation with ultraviolet rays in the range of 250 to 270 nm. This water-insoluble cross-linked product of single-stranded DNA or RNA also has the same action as the double-stranded DNA cross-linked product with respect to the above metals. It is also an object of the present invention to form a crosslinked body by irradiating a mixture of single-stranded and double-stranded DNA on a support with ultraviolet rays in the range of 250 to 270 nm.

The present invention will be described more specifically with reference to the following examples.
Example 1. Synthesis of cross-linked polymer of water-soluble DNA 10 mg of double-stranded DNA (molecular weight, 5 × 10 ⁶ ) obtained from salmon roe was dissolved in 1 mL of water, and the obtained DNA solution was poured into a petri dish having a diameter of 3 cm. Air dried at room temperature. Next, ultraviolet lamp (R-52G type, 5600 μW / cm ² , manufactured by Ultra Violet)
Was irradiated with ultraviolet rays of 254 nm (irradiation distance was about 7.5 cm) for 1 to 12 hours. Water was poured into the petri dish, and the water-insoluble DNA film formed on the petri dish bottom was peeled off from the petri dish bottom.

This water-insoluble DNA film was stable in water because it was insoluble in water even when immersed in water for a long period of 50 days. The water-insoluble DNA obtained by the ultraviolet irradiation was dissolved in boiling water at 100 ° C. in about 15 minutes. This dissolved sample is diluted with water to make a solution with a concentration of 0.1 μg / 10 μL, and then agarose gel electrophoresis (manufactured by Washina Sheng (manufactured), mobile phase TAE buffer, analysis temperature 30 ° C., moving distance 12 cm) Analysis showed that it was a very large compound with very high molecular weight. In addition, it has been confirmed that a compound having a very high molecular weight that does not migrate by agarose gel electrophoresis is also mixed in a water extract (reaction filtrate) of water-insoluble DNA at room temperature.

  Considering the high molecular weight that cannot be measured as water-insoluble at room temperature (25 ° C.), this water-insoluble DNA is a DNA cross-linked polymer produced by a cross-linking reaction of a large number of DNAs.

Example 2 Insertion with the above water-insoluble DNA film (film-like DNA cross-linked polymer)
Accumulation and removal of intercalating agent 1 mg (area 1 cm ² ) of the water-insoluble DNA film obtained in Example 1 was immersed in 10 mL of water containing the following intercalating agent at the following concentration to obtain a concentration in water: Was monitored by UV-VS spectroscopic absorbance at a given elapsed time and followed: ethidium bromide, 4 ppm; benzo [a] pyrene, 10 ppb; dibenzo-p-dioxane, 10 ppb; dibenzofuran, 10 ppb; biphenyl, 10 ppb. For example, in the case of ethidium bromide, the absorbance at the maximum absorption wavelength of 480 nm of the compound decreased with time, and disappeared completely in 24 hours. The same was observed for other intercalating agents.

Example 3 Immobilization of DNA on Cellulose Fiber Nonwoven Fiber by UV Irradiation 100 mg of cellulose fiber nonwoven fabric (cloth thickness 0.15 mm) was impregnated in a solution of 3 mg of DNA used in Example 1 in 1 mL of water. The impregnated nonwoven cellulose fibers were air-dried and irradiated with ultraviolet rays for a predetermined time in the same manner as in Example 1. Thereafter, the DNA not fixed was removed by washing thoroughly with water at about 20 ° C. and dried. The amount of DNA immobilized on the non-woven cellulose fibers generally increased with the time of ultraviolet irradiation, and reached a certain value after 15 minutes of irradiation. The maximum amount of DNA immobilized on 1 g of non-woven cellulose fiber was about 20 mg (maximum amount measuring method: 100 ° C., heated for 1 hr with 1N HCl, cooled to room temperature, 260 n
m).

Example 4 Obtained in Example 3 DNA fixed cellulose to the fiber nonwoven Ag ⁺ adsorption and Ag ⁺ DNA obtained in bactericidal activity Example 3 of adsorbed nonwoven fixed cellulosic fibrous nonwoven 30mg of (DNA content 20mg / cloth 1 g), Immerse in an aqueous solution containing 10 mM silver nitrate for 1 hour.
Next, the DNA-immobilized nonwoven fabric is taken out from the aqueous silver nitrate solution, washed with water, and dried at room temperature. The amount of Ag ⁺ adsorbed on the DNA-immobilized non-woven fabric after sufficient washing depends on the amount of silver nitrate used, and the amount of adsorption increases generally when the amount of silver nitrate is large. The maximum amount of Ag ⁺ adsorbed to 1 g of the DNA-immobilized nonwoven fabric was 5 mg.

The Ag ⁺ -containing DNA-immobilized non-woven fabric showed remarkable growth inhibitory properties against bacteria such as Escherichia coli and Staphylococcus aureus . This fabric can be widely used as an antimicrobial material.

  When a thin layer of water-soluble DNA or water-soluble RNA on a support or an aqueous solution of water-soluble DNA or water-soluble RNA or a liquid film thereof is irradiated with ultraviolet rays having a wavelength in the range of 250 to 270 nm, the DNA or RNA becomes water. It turns into a crosslinked body of supermacromolecules that are insoluble in water. Similarly, a cellulose fiber nonwoven fabric coated with a water-insoluble crosslinked polymer of DNA is formed by, for example, irradiating an aqueous solution of water-soluble DNA soaked with a cellulose fiber nonwoven fabric with ultraviolet rays having a wavelength in the range of 250 to 270 nm. The In the same manner, when glass peas coated with water-soluble DNA were irradiated with the same ultraviolet rays, a water-insoluble DNA cross-linked polymer was fixed on the surface. When this DNA is a double-stranded DNA, the cross-linked product adsorbs the intercalating agent for DNA or the environmental hormones that are intercalating agents for DNA even in a very low concentration state in water or in gas. Similarly, toxic metals such as Hg ions are also fixedly adsorbed to water. Therefore, this crosslinked product is useful as an environmental purification material that accumulates and removes environmental hormones and harmful metals. Specifically, air, water, drinking water, a filtering material for beverage food, or a cleaning agent in the digestive tract of mammals including humans, that is, pharmaceuticals, veterinary medicines or It can be used as an active ingredient contained in food. A single-stranded DNA or RNA cross-linked product is useful as an environmental purification material because it fixes harmful metals.

Claims (2)

A gas filter material for use in a tobacco filter or an air purifier for removing an intercalating agent into double-stranded DNA in the air or tobacco smoke, characterized by containing double-stranded DNA. 2. The gas filter material according to claim 1, wherein the intercalating agent into the double-stranded DNA is benzo [a] pyrene, benzo-p-dioxane, dibenzofuran and biphenyl.