JP2005278444A - Dna as functional material, method for preparing the same, and method for preparing highly functional dna film using the same - Google Patents
Dna as functional material, method for preparing the same, and method for preparing highly functional dna film using the same Download PDFInfo
- Publication number
- JP2005278444A JP2005278444A JP2004094775A JP2004094775A JP2005278444A JP 2005278444 A JP2005278444 A JP 2005278444A JP 2004094775 A JP2004094775 A JP 2004094775A JP 2004094775 A JP2004094775 A JP 2004094775A JP 2005278444 A JP2005278444 A JP 2005278444A
- Authority
- JP
- Japan
- Prior art keywords
- dna
- stranded
- stranded dna
- enzyme
- molecular weight
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
Description
近年DNAは、単に抵抗力の増加、老化防止などの生理機能に着目した健康食品として利用されるだけではなく、有機導電性素材、非線型光学材料、記録素子、有機EL素子、有害物質除去剤、医療用素材等の高分子機能性素材として注目されている。このように新しい機能性素材としてのDNAが、広く利用される為には、高純度のDNAであって、DNA特有の二重らせん、二本鎖構造を保持していること、分子量が揃っていること、一本鎖DNAが出来るだけ少ないことが必要とされている。本発明は、このような要請に応えた機能性素材としてのDNAを実現すべく、分子量を揃え、二本鎖を保持した高純度DNAを調製する方法と、機能性素材として望ましい姿に調製したDNAを提供せんとするものである。 In recent years, DNA is not only used as a health food focusing on physiological functions such as increased resistance and anti-aging, but also organic conductive materials, non-linear optical materials, recording elements, organic EL elements, harmful substance removal agents It is attracting attention as a functional polymer material such as medical materials. In order for DNA as a new functional material to be widely used in this way, it must be highly pure DNA, have a DNA-specific double helix and double-stranded structure, and have the same molecular weight. It is necessary to have as little single-stranded DNA as possible. In order to realize DNA as a functional material in response to such a request, the present invention was prepared in a desirable manner as a functional material and a method for preparing high-purity DNA with the same molecular weight and retaining double strands. It is intended to provide DNA.
DNAは、構造上安定で規則正しい二本鎖構造をしており、その塩基配列により、ｍRNAを経てペプチド合成に至る遺伝子情報を担う分子として良く知られている。一方、DNAは、その二本鎖の内側には、π電位に富んだ塩基が配列されており、電気を通しやすい性質や、分子を配向化する性質を持っている。また、その二本鎖中に平面構造の有害物質（ダイオキシンなど）を取り込むこと（インターカレート）ができる性質もある。このような特性から、ＤＮＡはその導電性を利用した導電性素材や非線型光学材料、インターカレーター能を利用した記録素子や有機ＥＬ素子、有害物質除去剤、医療用素材など様々な用途での利用が、期待されている。 DNA has a double-stranded structure that is stable and regular in structure, and is well known as a molecule that carries genetic information from mRNA to peptide synthesis through its base sequence. On the other hand, DNA has bases rich in π potential arranged inside the double strand, and has the property of easily conducting electricity and the property of orienting molecules. In addition, it has the property of being able to incorporate (intercalate) planar harmful substances (such as dioxin) into its double strand. Because of these characteristics, DNA is used in various applications such as conductive materials and non-linear optical materials that use its conductivity, recording elements and organic EL devices that use intercalator capabilities, harmful substance removers, and medical materials. Use is expected.
本来、ＤＮＡは、二本のポリヌクレオチドがらせんを巻いた二本鎖構造をもち、巨大な分子量を持った繊維状の形態を示している。この巨大な分子量を持つＤＮＡは機能性素材等として期待されているが、分子量が大きいほど溶媒への溶解が困難であり、扱い難い問題を持ち合わせている。そのため、この巨大な分子量を持った高分子ＤＮＡを分解して、適当な分子量のＤＮＡに低分子化することにより、取扱い易くなり、機能性素材として、用途の拡大が見込まれる。 Originally, DNA has a double-stranded structure in which two polynucleotides are wound in a helix, and shows a fibrous form having a huge molecular weight. This DNA having a huge molecular weight is expected as a functional material and the like. However, the larger the molecular weight, the more difficult it is to dissolve in a solvent, and it is difficult to handle. Therefore, it is easy to handle by decomposing high molecular weight DNA having a large molecular weight and reducing the molecular weight to a DNA having an appropriate molecular weight, and the use of the functional material is expected to expand.
従来より行われているＤＮＡの製造方法では、加熱や酸や酵素により二本鎖構造の破壊や低分子化が起こることが知られている。しかし、ＤＮＡの製造法においてその二本鎖構造の破壊や低分子化をコントロールすることまでは、発想されておらず、二本鎖のＤＮＡと一本鎖のＤＮＡと、大きさの異なる分子量のＤＮＡとが広く混在した粉末になっているのが普通である。しかし、このように、二本鎖構造が破壊されてしまっていては、機能性素材としてのＤＮＡの機能や特性が失われてしまうことが多く、また分子量の範囲が広いとその分子量の違いによって、粘性等の物性が異なり、製造等において取扱い難いものとなる。 In conventional DNA production methods, it is known that the double-stranded structure is broken or the molecular weight is reduced by heating, acid or enzyme. However, it has not been conceived until the destruction of the double-stranded structure and the reduction in molecular weight are controlled in the production method of DNA, and double-stranded DNA and single-stranded DNA have different molecular weights. The powder is usually mixed with DNA widely. However, if the double-stranded structure is destroyed in this way, the function and properties of DNA as a functional material are often lost, and if the molecular weight range is wide, , The physical properties such as viscosity are different, making it difficult to handle in production.
そのため、機能性素材として役立つＤＮＡは、少なくとも次のような条件を備えていることが望まれている。
ａ）夾雑タンパクの少ない高純度ＤＮＡであること。
ｂ）二重らせん、二本鎖構造を保持しているＤＮＡであること。
ｃ）一本鎖DNAを出来るだけ除去し、二本鎖率の高いＤＮＡになっていること。
ｄ）適度な大きさの分子量に揃っているＤＮＡであること。
Therefore, it is desired that DNA useful as a functional material has at least the following conditions.
a) High purity DNA with few contaminating proteins.
b) DNA having a double helix or double strand structure.
c) The single-stranded DNA is removed as much as possible, and the DNA has a high double-stranded rate.
d) DNA having an appropriate molecular weight.
尚、機能性素材としてのＤＮＡは、高分子であればあるほど、溶液時の粘性が高くなり扱いが困難になることから、ある程度低分子化し、粘性を低下させることも求められる。即ち、用途や目的によっては分子量が所定の大きさに揃っていることが要請されている。従って、使い勝手のよい機能性素材等としてのＤＮＡとは、ＤＮＡを適度に低分子化処理し、粘性を低下させたものといえるが、同時に目的や用途に応じて分子量を揃えるようにすることと、二本鎖が保持されていることという調製がなされたＤＮＡということが出来る。 In addition, since the DNA as a functional material has a higher viscosity at the time of solution and the handling becomes more difficult as the polymer is higher, it is also required to lower the molecular weight to some extent and lower the viscosity. That is, depending on the application and purpose, it is required that the molecular weights have a predetermined size. Therefore, DNA as an easy-to-use functional material can be said to have been obtained by appropriately reducing the molecular weight of the DNA to lower the viscosity, but at the same time, adjusting the molecular weight according to the purpose and application. It can be said that the DNA has been prepared such that the double strand is retained.
二本鎖構造を保持した高分子ＤＮＡの大量精製技術については、近年各方面で研究開発が進められているが、いまだ工業的規模での生産が行われる段階には至っていない。そのため、二本鎖構造を保持した高分子ＤＮＡは非常に高価である。このような背景から、本発明者らは、機能性素材として様々な分野からのニーズに答えられる使い勝手の良い、機能性素材としてのＤＮＡを製造することを目的に、二本鎖を保持し、望ましい所定の分子量が揃ったＤＮＡを調製する技術を開発することとした。 In recent years, research and development has been progressed in various fields regarding a large-scale purification technique for polymer DNA having a double-stranded structure, but it has not yet reached the stage of production on an industrial scale. For this reason, a high molecular weight DNA having a double-stranded structure is very expensive. From such a background, the present inventors maintain a double strand for the purpose of producing DNA as a functional material that is easy to use and can meet the needs from various fields as a functional material, It was decided to develop a technique for preparing DNA having a desired predetermined molecular weight.
本出願人は、これまでにサケ白子由来のヌクレオプロテインからドデシル硫酸ナトリウム（ＳＤＳ）を用いた抽出・精製により、高純度でかつ高分子量のＤＮＡを大量に調製する方法を種々研究開発し確立してきた。その調製方法の一例が、図１に示した工程図の通りである。この調製法は、先ず、本出願人において、サケ白子から調製したヌクレオプロテインを原料として用いた。当該ヌクレオプロテインを水に懸濁し、ＳＤＳおよびNａＣｌを添加してＤＮＡを抽出した。ＳＤＳとコンプレックスを形成したプロタミンを遠心分離により除去し、得られた上清にエタノール（EｔOH）を加えてＤＮＡを沈殿物として得た。得られたＤＮＡを洗浄後、凍結乾燥により乾燥し、白色で繊維状のDNAを得た。更に、本出願人は、コスト低減の検討に関して、エタノールに代えて変性アルコール等の使用及びＵＦ膜を用いる方法を開発した。 The applicant has so far researched and developed various methods for preparing large amounts of high-purity and high-molecular-weight DNA from extraction and purification using sodium dodecyl sulfate (SDS) from nucleoprotein derived from salmon eggplant. It was. An example of the preparation method is as shown in the process diagram of FIG. In this preparation method, first, the present applicant used nucleoprotein prepared from salmon milk as a raw material. The nucleoprotein was suspended in water, and SDS and NaCl were added to extract DNA. Protamine complexed with SDS was removed by centrifugation, and ethanol (EtOH) was added to the resulting supernatant to obtain DNA as a precipitate. The obtained DNA was washed and then dried by lyophilization to obtain white and fibrous DNA. Furthermore, the present applicant has developed a method using a denatured alcohol or the like and a UF membrane in place of ethanol with regard to cost reduction studies.
このような図1の方法で得られたＤＮＡは、市販されているサケ由来のＤＮＡ（Ｓｉｇｍａ社製；二本鎖率約８０％）と比較してＤＮＡ含量が同等であり、また夾雑タンパク含量が、１％未満と、市販品より優れていた。そのＤＮＡの分析結果は、表１の通りである。この結果、本製法によって市販品と同等以上のタンパク含量の低い、高純度のＤＮＡが得られた。しかも、本製法で製造された高純度ＤＮＡは、ＮａＣｌを用いて、温和な条件でＤＮＡを抽出しているので高分子量で二本鎖構造を保持したＤＮＡが高収率で含まれている。従って、この製法で得られた高純度で高分子量のＤＮＡは、機能性素材としてのＤＮＡを調製する原料としてふさわしいと判断された。 The DNA obtained by the method of FIG. 1 has a DNA content equivalent to that of a commercially available salmon-derived DNA (manufactured by Sigma; double-strand rate of about 80%), and a contaminating protein content. However, it was less than 1% and better than the commercial product. The analysis results of the DNA are shown in Table 1. As a result, high purity DNA having a protein content equal to or lower than that of a commercially available product was obtained by this production method. Moreover, since the high-purity DNA produced by this production method is extracted using NaCl under mild conditions, it contains a high-molecular-weight DNA having a double-stranded structure in a high yield. Therefore, the high purity and high molecular weight DNA obtained by this production method was judged to be suitable as a raw material for preparing DNA as a functional material.
本発明者らは、前記製法で得られた高純度高分子ＤＮＡを原料として用いて、機能性素材等としてのＤＮＡを調製することを目的とした。尚、原料となる高純度高分子ＤＮＡというのは、二本鎖構造を保持しておりかつ高純度高分子であれば、必ずしも前記の調製法により得られたものに限る必要がなく、他の方法で調製されたものであっても良いことは勿論である。 The present inventors have aimed to prepare DNA as a functional material or the like using the high purity polymer DNA obtained by the above production method as a raw material. The high-purity polymer DNA used as a raw material is not necessarily limited to that obtained by the above preparation method as long as it has a double-stranded structure and is a high-purity polymer. Of course, it may be prepared by a method.
高分子ＤＮＡを分解して低分子化し、所定の分子量に調整する技術としては超音波処理や酸、アルカリ処理が考えられるがＤＮＡの二本鎖構造が破壊される等あまり実用的ではない。その点、ＤＮＡ分解能を有する酵素は、種々ありその分解能特性も様々である。その点に着目して、酵素によるＤＮＡの分解処理について研究することとした。 Ultrasonic treatment, acid treatment, and alkali treatment are conceivable as techniques for degrading high molecular DNA to lower the molecular weight and adjusting the molecular weight to a predetermined molecular weight, but it is not very practical because the double-stranded structure of DNA is destroyed. In that respect, there are various enzymes having DNA resolution, and the resolution characteristics are also various. Focusing on this point, we decided to study DNA degradation treatment with enzymes.
本発明者らは、鋭意研究の結果、高純度高分子ＤＮＡをＤＮＡ鎖を切断する酵素を用いて酵素処理すると、まず、所定の大きさに揃った分子量に低分子化できること、及びＤＮＡ一本鎖ＤＮＡを特異的に分解する酵素によって酵素処理すると、一本鎖ＤＮＡを切断・除去することができるとの知見を得た。そこで、両酵素による処理を組み合わせてみたところ、分子量を調整しながら二本鎖率の高いＤＮＡを調製できることを見出し、本発明を完成させたものである。 As a result of diligent research, the inventors of the present invention have found that when high-purity polymer DNA is enzymatically treated with an enzyme that cleaves a DNA strand, first, the molecular weight can be reduced to a predetermined molecular weight, and a single DNA can be obtained. It was found that single-stranded DNA can be cleaved and removed by enzymatic treatment with an enzyme that specifically degrades the strand DNA. Thus, when the treatments with both enzymes were combined, it was found that DNA having a high double-stranded rate could be prepared while adjusting the molecular weight, and the present invention was completed.
特許を受けようとする第１発明は、高純度高分子ＤＮＡに、ＤＮＡ鎖を切断し得る酵素を加えて酵素処理し、目的に応じた分子量のＤＮＡに切断調整した後、これに一本鎖ＤＮＡを特異的に切断し得る酵素を加えて更に酵素処理して一本鎖ＤＮＡを切断し、その切断された一本鎖ＤＮＡを除去することによって、目的の分子量に揃えるとともに二本鎖ＤＮＡの割合が高くなることを特徴とする機能性素材としてのＤＮＡの調製方法である。 The first invention to be patented is to add an enzyme capable of cleaving a DNA strand to high-purity high-molecular DNA, and then perform enzymatic treatment to prepare a DNA having a molecular weight according to the purpose. By adding an enzyme capable of specifically cleaving the DNA and further treating with an enzyme, the single-stranded DNA is cleaved, and the cleaved single-stranded DNA is removed. This is a method for preparing DNA as a functional material characterized by a high ratio.
本発明に用いられるＤＮＡ鎖を切断し得る酵素とは、高分子のＤＮＡを目的の分子量に調整するためにＤＮＡ鎖を切断する分解能を有する酵素であり試薬レベルにまで精製されていることに限らず、工業用酵素等でもＤＮＡ鎖切断能を有すればこれに該当する。これらの例としてはデオキシリボヌクレアーゼＩ、デオキシリボヌクレアーゼＩＩ、ホスホジエステラーゼ、Ｍｉｃｒｏｃｏｃｃａｌヌクレアーゼ、また、食品工業用酵素ではウマミザイム、ニューラーゼＦ、プロテアーゼＡ、プロテアーゼＭ、プロテアーゼＰ３、プロメラインＦ、プロレザー（以上、天野エンザイム（株）製）、パンチダーゼＮＰ−2、プロテアーゼＹＰ−ＳＳ（以上、ヤクルト薬品工業（株）製）、フレーバーザイム（ノボノルディスク製）、精製パパインＦ（アサヒビール（株）製）、ビオプラーゼＰＮ−4（ナガセケムテックス（株）製）等が挙げられる。 The enzyme capable of cleaving a DNA strand used in the present invention is an enzyme having a resolution capable of cleaving a DNA strand in order to adjust high molecular DNA to a target molecular weight, and is limited to being purified to a reagent level. In addition, an industrial enzyme or the like corresponds to this if it has a DNA strand-cleaving ability. Examples of these include deoxyribonuclease I, deoxyribonuclease II, phosphodiesterase, Micrococcal nuclease, and as enzymes for food industry, equinezyme, newase F, protease A, protease M, protease P3, promelain F, proleather (above, Amano) Enzyme Co., Ltd.), Pandidase NP-2, Protease YP-SS (Yakult Pharmaceutical Co., Ltd.), Flavorzyme (Novo Nordisk), Purified Papain F (Asahi Beer Co., Ltd.), Bioprase PN-4 (manufactured by Nagase ChemteX Corporation) and the like.
本発明に用いられるＤＮＡ一本鎖を特異的に切断する酵素とは、特に限定されるものでなく、例えばBAL 31 ヌクレアーゼ、Ｓ１ヌクレア−ゼ、マングマメヌクレア−ゼ（ヌクレア−ゼＭＢ）、ヌクレアーゼＰ１等が挙げられる。 The enzyme that specifically cleaves a DNA single strand used in the present invention is not particularly limited, and examples thereof include BAL 31 nuclease, S1 nuclease, mung bean nuclease (nuclease MB), Examples include nuclease P1.
前記二種類の分解能特性をもった酵素による酵素処理を組み合せることにより、目的の分子量に揃え且つ二本鎖ＤＮＡの割合が高い機能性素材としてのＤＮＡを調製できるのである。 By combining enzyme treatments with enzymes having the two types of resolution characteristics, it is possible to prepare DNA as a functional material that has the same molecular weight and a high proportion of double-stranded DNA.
特許を受けようとする第２発明は、高純度高分子ＤＮＡに、ＤＮＡ鎖を切断し得る酵素を加えて酵素分解処理し、目的に応じて分子量５万−３００万（１００−５０００塩基対）のＤＮＡに切断調整した後、これに一本鎖ＤＮＡを特異的に切断し得る酵素を加えて酵素処理して一本鎖ＤＮＡを切断し、その切断された一本鎖ＤＮＡを除去することによって、目的に応じて分子量５万−３００万（１００−５０００塩基対）に揃えるとともに二本鎖ＤＮＡの割合が高くなることを特徴とする機能性素材としてのＤＮＡの調製方法である。 The second invention to be patented is a high-purity polymer DNA, which is subjected to an enzymatic degradation treatment by adding an enzyme capable of cleaving the DNA strand, and a molecular weight of 50,000 to 3 million (100-5000 base pairs) depending on the purpose. After adjusting the cleavage of the DNA, an enzyme capable of specifically cleaving the single-stranded DNA is added to the DNA, and the enzyme treatment is performed to cleave the single-stranded DNA, and the cleaved single-stranded DNA is removed. This is a method for preparing DNA as a functional material characterized by having a molecular weight of 50,000 to 3 million (100-5000 base pairs) according to the purpose and increasing the proportion of double-stranded DNA.
当該第２発明の分子量を揃える範囲については、酵素のＤＮＡ分解能と、本発明を実施する目的とを勘案して実用性、実現性の可能なものとして確認して、分子量５万−３００万（１００−５０００塩基対）の範囲に限定した。尚、今後の研究如何では更に高分子のＤＮＡや低分子のＤＮＡが求められる可能性があるが、この製法を使うと上記範囲外であっても要求される分子量のＤＮＡを調製することが可能と思われる。 About the range which arrange | equalizes the molecular weight of the said 2nd invention, considering the DNA resolution of an enzyme and the objective which implements this invention, it confirmed that it was practicable and feasible, molecular weight 50,000-3 million ( 100-5000 base pairs). Depending on the future research, higher molecular weight DNA or lower molecular weight DNA may be required, but if this method is used, DNA with the required molecular weight can be prepared even outside the above range. I think that the.
特許を受けようとする第３発明は、高純度高分子ＤＮＡに、ＤＮＡ鎖を切断し得る酵素を加えて酵素分解処理することにより目的に応じた所定の分子量のＤＮＡに切断調整したうえ、これに一本鎖ＤＮＡを特異的に切断し得る酵素を加えて酵素処理することにより一本鎖ＤＮＡを切断し、その切断された一本鎖ＤＮＡを除去することによって、目的に応じて分子量を５万−３００万（１００−５０００塩基対）に揃えるとともに、二本鎖ＤＮＡの割合が高くなるようにして、二本鎖構造に基づくDNAの機能が高められたことを特徴とする機能性素材としてのＤＮＡである。 The third invention to be patented is a high-purity polymer DNA, which is prepared by adding an enzyme capable of cleaving the DNA strand and subjecting it to enzymatic degradation, thereby adjusting the cleavage to DNA of a predetermined molecular weight according to the purpose. In addition, an enzyme capable of specifically cleaving single-stranded DNA is added and treated with an enzyme to cleave the single-stranded DNA, and the cleaved single-stranded DNA is removed. As a functional material characterized in that the function of DNA based on the double-stranded structure is enhanced by aligning it to 10,000 to 3,000,000 (100-5000 base pairs) and increasing the proportion of double-stranded DNA DNA.
当該第３発明は、機能性素材としてのＤＮＡという物の発明である。近年、機能性素材としてのＤＮＡが注目されるようになり、各方面で研究されているが、目的に応じて分子量を揃えるように切断することと、一本鎖ＤＮＡを切断し、その切断された一本鎖ＤＮＡを可及的に除去して二本鎖ＤＮＡの割合を高くするという２つの要請を同時に満足させることは、技術的に困難な課題であるとされていた。このように機能性素材としてのＤＮＡに前記のような２つの要請を同時に満足させることは、二本鎖ＤＮＡの構造を出来るだけ壊すことなく、取り扱いやすい分子量に切断することである。特に一本鎖ＤＮＡを特異的に切断し、その切断された一本鎖ＤＮＡを可及的に除去することは、これまで想起されておらず、それを実現する技術の提案はされていなかった。本発明者は、高分子ＤＮＡを酵素処理によって切断する方法に着目し、鋭意研究をしたところ、酵素の種類、酵素処理の際の温度や濃度や時間などの条件を調整することによって、切断する分子量の大きさも、一本鎖ＤＮＡの特異的な切断についてもコントロールしながら調製することが可能であるとの新しい知見を得た。本発明は、この新しい技術的知見に基づいて本願発明を完成したものである。その結果、目的に応じて分子量が揃い、二本鎖ＤＮＡの割合が高くて、二本鎖構造に基づくDNAの機能が高められたことを特徴とする機能性素材としてのＤＮＡを初めて具現化することができたのである。 The third invention is an invention of DNA as a functional material. In recent years, DNA as a functional material has attracted attention and has been studied in various fields. However, it has been studied to cut the single-stranded DNA by cutting it so that the molecular weights are aligned according to the purpose. In addition, it has been regarded as a technically difficult problem to simultaneously satisfy the two requirements of removing single-stranded DNA as much as possible to increase the ratio of double-stranded DNA. Thus, satisfying the above two requirements at the same time for DNA as a functional material is to cut the molecular weight of the double-stranded DNA into an easy-to-handle molecular weight as much as possible. In particular, it has not been conceived so far to specifically cleave single-stranded DNA and to remove the cleaved single-stranded DNA as much as possible, and no technology has been proposed to realize it. . The present inventor paid attention to a method for cleaving high-molecular DNA by enzyme treatment and conducted extensive research, and cleaved by adjusting conditions such as the type of enzyme, temperature, concentration, and time during enzyme treatment. We obtained new knowledge that the molecular weight can be adjusted while controlling the specific cleavage of single-stranded DNA. The present invention has been completed based on this new technical knowledge. As a result, DNA as a functional material is realized for the first time, which is characterized by the fact that the molecular weight is aligned according to the purpose, the percentage of double-stranded DNA is high, and the function of DNA based on the double-stranded structure is enhanced. It was possible.
特許を受けようとする第４発明は、高純度高分子ＤＮＡに、ＤＮＡ鎖を切断し得る酵素としてプロテアーゼＰ３を加えて酵素分解処理し、ほぼ２００−６００塩基対のＤＮＡに切断調整した後、これにＳ１ヌクレア−ゼを加えて酵素処理して一本鎖ＤＮＡを切断し、その切断された一本鎖ＤＮＡを除去することによって、目的に応じて３００−５００塩基対に揃えるとともに二本鎖ＤＮＡの割合が高くなることを特徴とする機能性素材としてのＤＮＡの調製方法である。 According to a fourth invention to be patented, high-purity polymer DNA is subjected to an enzymatic degradation treatment by adding protease P3 as an enzyme capable of cleaving a DNA strand, and after cleaving to approximately 200-600 base pairs of DNA, By adding S1 nuclease to this and treating it with an enzyme to cleave the single-stranded DNA and remove the cleaved single-stranded DNA, it is aligned to 300-500 base pairs according to the purpose and double-stranded. This is a method for preparing DNA as a functional material, characterized in that the proportion of DNA increases.
当該第４発明は、ＤＮＡ鎖を切断し得る酵素としてプロテアーゼＰ３、S１ヌクレアーゼを用いて、３００−５００塩基対に揃えるとともに二本鎖ＤＮＡの割合が高いＤＮＡに調製した実施態様項である。当該特定された分子量の範囲は、目的の汎用性が広いうえ、粘性も適度に低下していて使い勝手のよい機能性素材としてのＤＮＡとなっている。 The fourth invention is an embodiment in which protease P3, S1 nuclease is used as an enzyme capable of cleaving a DNA strand, and the DNA is adjusted to 300-500 base pairs and has a high proportion of double-stranded DNA. The specified molecular weight range is DNA as a functional material that is easy to use and has a wide range of target versatility and a moderately reduced viscosity.
特許を受けようとする第５発明は、高純度高分子ＤＮＡに、ＤＮＡ鎖を切断し得る酵素を加えて酵素処理し、目的に応じた分子量のＤＮＡに切断調整した後、これに一本鎖ＤＮＡを特異的に切断し得る酵素を加えて更に酵素処理して一本鎖ＤＮＡを切断し、その切断された一本鎖ＤＮＡを除去することによって、目的の分子量に揃えるとともに二本鎖ＤＮＡの割合が高くなることを特徴とする機能性素材としてのＤＮＡに調製したうえ、当該目的の分子量に調整し且つ二本鎖ＤＮＡの割合が高い機能性素材としてのＤＮＡ溶液と脂質溶液とを混合攪拌してＤＮＡ−脂質コンプレックスを得て、当該ＤＮＡ−脂質コンプレックスを有機溶媒に溶解し、これを薄膜状に延ばして乾燥させ、薄膜状のＤＮＡフイルムを得るようにしたことを特徴とする高機能性ＤＮＡフイルムの調製方法である。 According to a fifth invention to be patented, an enzyme capable of cleaving a DNA strand is added to high-purity high-molecular DNA, treated with an enzyme, adjusted to cleave to a molecular weight DNA according to the purpose, and then single-stranded into this. By adding an enzyme capable of specifically cleaving the DNA and further treating with an enzyme, the single-stranded DNA is cleaved, and the cleaved single-stranded DNA is removed. Prepared as DNA as a functional material characterized by a high ratio, mixed and stirred the DNA solution and lipid solution as a functional material adjusted to the target molecular weight and with a high percentage of double-stranded DNA To obtain a DNA-lipid complex, and the DNA-lipid complex is dissolved in an organic solvent, which is stretched into a thin film and dried to obtain a thin-film DNA film. It is a process for the preparation of highly functional DNA film.
当該第５発明は、機能性素材としてのＤＮＡを利用した高機能性ＤＮＡフイルムの調製方法である。ＤＮＡフイルムであって目的に応じて分子量を揃えるとともに二本鎖ＤＮＡの割合が高いものは、高機能性を具備しているが、その製造が困難である。本発明のように前段で、酵素を利用して分解処理し、目的の分子量に揃えるとともに二本鎖ＤＮＡの割合が高い機能性素材としてのＤＮＡとなし、次いで当該調製された機能性素材としてのＤＮＡを原料として薄膜化加工処理をすることによって簡単に高機能性ＤＮＡフイルムを調製することができる。このように、本願発明を用いれば高機能性ＤＮＡフイルムを工業的手段で大量生産することが可能となる。 The fifth invention is a method for preparing a highly functional DNA film using DNA as a functional material. A DNA film having a uniform molecular weight according to the purpose and having a high percentage of double-stranded DNA has high functionality but is difficult to produce. As in the present invention, in the previous stage, degradation is performed using an enzyme, the DNA is made into a functional material that has the same molecular weight and has a high percentage of double-stranded DNA, and then the prepared functional material. A highly functional DNA film can be easily prepared by performing a thinning process using DNA as a raw material. As described above, the use of the present invention makes it possible to mass-produce highly functional DNA films by industrial means.
特許を受けようとする第６発明は、高純度高分子ＤＮＡに鎖を切断し得る酵素プロテアーゼＰ３を加えて酵素処理し、ほぼ２００−６００塩基対のＤＮＡに切断調整した後、これに一本鎖ＤＮＡを特異的に切断し得る酵素としてＳ１ヌクレア−ゼを加えて酵素処理して一本鎖ＤＮＡを切断し、その切断された一本鎖ＤＮＡを除去することによって、３００−５００塩基対に揃えるとともに二本鎖ＤＮＡの割合が高くなっている機能性素材としてのＤＮＡに調製する。そのうえで、当該３００−５００塩基対に調整し且つ二本鎖ＤＮＡの割合が高い機能性素材としてのＤＮＡ溶液と脂質溶液とを混合攪拌してＤＮＡ−脂質コンプレックスを得て、当該ＤＮＡ−脂質コンプレックスを熱による圧着や有機溶媒に溶解し、これを広げるように延ばして乾燥させ、薄膜状のＤＮＡフイルムを得るようにしたことを特徴とする高機能性ＤＮＡフイルムの調製方法である。 According to the sixth invention to be patented, enzyme protease P3 capable of cleaving a strand is added to high-purity polymer DNA and subjected to enzyme treatment to adjust the cleavage to about 200-600 base pairs of DNA. By adding S1 nuclease as an enzyme capable of specifically cleaving strand DNA and treating with enzyme to cleave the single-stranded DNA, and removing the cleaved single-stranded DNA, 300-500 base pairs are obtained. The DNA is prepared as a functional material having a high double-stranded DNA ratio. Then, a DNA solution and a lipid solution as a functional material adjusted to 300-500 base pairs and a high proportion of double-stranded DNA are mixed and stirred to obtain a DNA-lipid complex. This is a method for preparing a highly functional DNA film, characterized in that a thin film-like DNA film is obtained by heat-bonding or dissolving in an organic solvent, spreading the film and drying it.
当該第６発明は、使い勝手のよい機能性素材としてのＤＮＡを用いて実用性の高い高機能性ＤＮＡフイルムの調製方法を具現化した実施態様項である。 The sixth invention is an embodiment in which a method for preparing a highly functional DNA film having high practicality is realized using DNA as a functional material that is easy to use.
本願発明は、分子量を調整した二本鎖ＤＮＡの収率が高い機能性素材としてのＤＮＡや高機能性ＤＮＡフイルムの調製方法である。まず、機能性素材としてのＤＮＡは様々な分野での利用が考えられているが、その用途によって求められているＤＮＡの分子量は異なっている。しかし、どの分野でも重要なのは常に二本鎖を保持していることである。このように、目的の分子量に調製でき且つ二本鎖を保持しているDNAは、その機能性や品質が安定しているうえ、その粘度などの物性も一定であるため、取扱いやすいものとなり、機能性素材のＤＮＡとして高い評価を受けることができる。本発明は、この高い評価を受ける機能性素材としてのＤＮＡを所定の分子量に調整するための酵素と、一本鎖ＤＮＡを特異的に切断し、除去することによって二本鎖の割合を高めるための酵素との二種類の酵素処理により、簡単に調製できるようにしたものである。 The present invention is a method for preparing DNA or a high-functional DNA film as a functional material having a high yield of double-stranded DNA with an adjusted molecular weight. First, DNA as a functional material is considered to be used in various fields, but the required molecular weight of DNA varies depending on its use. However, what is important in any field is that it always retains double strands. In this way, DNA that can be prepared to the desired molecular weight and retains double strands has stable functionality and quality, and its physical properties such as viscosity are also constant, making it easy to handle, Highly evaluated as functional material DNA. The present invention increases the ratio of double strands by specifically cleaving and removing single-stranded DNA and an enzyme for adjusting DNA as a functional material that is highly evaluated to have a predetermined molecular weight. It can be easily prepared by two kinds of enzyme treatment with the above enzyme.
また、分子量を調整した二本鎖ＤＮＡの収率が高い機能性素材としてのＤＮＡを用いて調製する高機能性ＤＮＡフイルムは、ＤＮＡ一本鎖を特異的に切断する酵素を作用させ、二本鎖率を高めることにより、より低い濃度でフイルム化することができるようになった。尚、本発明に係る調製法で得られた高機能性ＤＮＡフイルムは、高分子ＤＮＡにより作製されたフイルムと比較すると、少し厚みのあるフイルムであるが、粘性がなく、扱いやすい利点がある。 In addition, a highly functional DNA film prepared by using DNA as a functional material having a high yield of double-stranded DNA with an adjusted molecular weight is an enzyme that specifically cleaves DNA single strands. By increasing the chain ratio, the film can be formed at a lower concentration. The highly functional DNA film obtained by the preparation method according to the present invention is a slightly thicker film than the film produced by polymer DNA, but has the advantage that it is not viscous and easy to handle.
本願発明について、以下、実施例に基づいて、詳細に説明する。 Hereinafter, the present invention will be described in detail based on examples.
＜二本鎖３００−５００塩基対ＤＮＡの調製方法＞
２．５ｍｇ／ｍｌの高純度高分子ＤＮＡ ８０ｍｌに、０．１ｍｇ／ｍｌのプロテアーゼＰ３（天野エンザイム（株）製）を１０ｍｌ加えて、４５℃で３０分間加熱した後、酵素反応を停止させ、回収した。このようにプロテアーゼＰ３を作用させることにより、その中の夾雑ヌクレア−ゼにより、低分子化することによって所定の分子量に調整されたＤＮＡとなった。その後、得られたＤＮＡ溶液に、Ｓ１ヌクレア−ゼ（３０００Ｕ）６００μｌを加えて３７℃で３０分間加熱した後、ＤＮＡを回収した。その結果、Ｓ１ヌクレア−ゼにより、分子量が２０−３０万の３００−５００塩基対とほぼ揃ったＤＮＡに低分子化するとともに、一本鎖ＤＮＡを特異的に切断・除去して、二本鎖率を高めることができた。当該二本鎖３００−５００塩基対ＤＮＡの調製方法のフローを図２に示す。
<Preparation method of double-stranded 300-500 base pair DNA>
10 ml of 0.1 mg / ml protease P3 (manufactured by Amano Enzyme) was added to 80 ml of 2.5 mg / ml high-purity polymer DNA and heated at 45 ° C. for 30 minutes, and then the enzyme reaction was stopped. It was collected. Thus, by making protease P3 act, it became DNA adjusted to the predetermined | prescribed molecular weight by making low molecular weight with the contaminating nuclease in it. Thereafter, 600 μl of S1 nuclease (3000 U) was added to the obtained DNA solution and heated at 37 ° C. for 30 minutes, and then DNA was recovered. As a result, the S1 nuclease reduces the molecular weight to a DNA having a molecular weight of 200-300,000 and approximately 300-500 base pairs, and also specifically cleaves and removes the single-stranded DNA. The rate could be increased. The flow of the method for preparing the double-stranded 300-500 base pair DNA is shown in FIG.
＜分子量の確認＞
前記調製方法により得られた二本鎖３００−５００塩基対ＤＮＡの分子量分布をキャピラリー電気泳動により調べた。その結果、図３に示すように二本鎖３００−５００塩基対ＤＮＡは大部分が目的の分子量である３００−５００塩基対DNA付近に分布していることが確認できた。
<Confirmation of molecular weight>
The molecular weight distribution of double-stranded 300-500 base pair DNA obtained by the above preparation method was examined by capillary electrophoresis. As a result, as shown in FIG. 3, it was confirmed that most of the double-stranded 300-500 base pair DNA was distributed in the vicinity of the target molecular weight of 300-500 base pair DNA.
＜ＤＮＡ二本鎖率の測定＞
次に、前記調製方法により得られた二本鎖３００−５００塩基対ＤＮＡをＳ１ヌクレア−ゼを用いたDNA二本鎖率測定法により測定した。その結果は、表２の通りＳ１ヌクレア−ゼ作用により、二本鎖率が高まることを確認した。
<Measurement of DNA double strand rate>
Next, the double-stranded 300-500 base pair DNA obtained by the above preparation method was measured by a DNA double-stranded ratio measuring method using S1 nuclease. As a result, as shown in Table 2, it was confirmed that the double-stranded rate was increased by the S1 nuclease action.
このように二本鎖３００−５００塩基対ＤＮＡ調製において、Ｓ１ヌクレア−ゼ処理によって二本鎖率は増加した。そして、このＳ１ヌクレア−ゼ処理によって二本鎖を９０％以上保持したＤＮＡが得られることが確認できた。これらのことから、高純度高分子ＤＮＡから、プロテアーゼＰ３中の夾雑ヌクレア−ゼにより低分子化した後、Ｓ１ヌクレア−ゼ処理により一本鎖部分を除去することにより、二本鎖３００−５００塩基対ＤＮＡを得ることが出来た。 Thus, in the preparation of double-stranded 300-500 base pairs DNA, the double-stranded rate increased by the S1 nuclease treatment. It was confirmed that DNA having 90% or more of the double strands was obtained by this S1 nuclease treatment. From these facts, double-stranded 300-500 bases are obtained by removing the single-stranded portion from the high-purity high-molecular-weight DNA with a contaminating nuclease in protease P3 and then treating with S1 nuclease. We were able to obtain anti-DNA.
＜二本鎖３００−５００塩基対DNA−脂質コンプレックスとそのフイルムの調製＞
二本鎖ＤＮＡの収率が高い機能性素材としてのＤＮＡとして５０ｍｇ／２０ｍｌ二本鎖３００−５００塩基対ＤＮＡ溶液と１．１倍当量の脂質溶液（ジラウリルジメチルアンモニウムブロミド）を混合し、室温で1時間攪拌した。そして未反応ＤＮＡの洗浄のため、脱イオン水添加と遠心分離（１００００ｒｐｍ、０℃、１０分）を３回繰り返した。その後、脱脂する目的でジエチルエーテルを添加し、遠心分離（１２０００ｒｐｍ、０℃、１０分）を３回繰り返した。その後、凍結乾燥し、ＤＮＡ−脂質コンプレックスを得た。
このＤＮＡ−脂質コンプレックスをクロロホルム・エタノール溶液（クロロホルム・エタノール＝４：１）に所定濃度になるように溶解させた。そしてクロロホルム・エタノール溶液を滴下したガラスシャーレ内にテフロンプレートを置き、そのテフロンプレート上に前記ＤＮＡ−脂質コンプレックス溶液を移した。１日静置後、乾燥したものをＤＮＡフイルムとして回収した。尚、ＤＮＡフイルムの調製法は岡畑らの方法（J．Am．Chem．Soc．，１１８（44），10679（1996）を元に行った。
<Preparation of double-stranded 300-500 base pair DNA-lipid complex and its film>
50 mg / 20 ml double-stranded 300-500 base pair DNA solution and 1.1-fold equivalent lipid solution (dilauryldimethylammonium bromide) are mixed as DNA as a functional material with high yield of double-stranded DNA, and room temperature For 1 hour. And in order to wash | clean unreacted DNA, deionized water addition and centrifugation (10000 rpm, 0 degreeC, 10 minutes) were repeated 3 times. Then, diethyl ether was added for the purpose of degreasing, and centrifugation (12000 rpm, 0 ° C., 10 minutes) was repeated three times. Thereafter, it was freeze-dried to obtain a DNA-lipid complex.
This DNA-lipid complex was dissolved in a chloroform / ethanol solution (chloroform / ethanol = 4: 1) to a predetermined concentration. Then, a Teflon plate was placed in a glass petri dish to which a chloroform / ethanol solution was dropped, and the DNA-lipid complex solution was transferred onto the Teflon plate. After standing for 1 day, the dried product was recovered as a DNA film. The DNA film was prepared based on the method of Okabata et al. (J. Am. Chem. Soc., 118 (44), 10679 (1996)).
Ｓ１ヌクレア−ゼを作用させたものとさせていないものの２種類を用い、フイルムの比較を行った。２００μｌのクロロホルム・エタノール溶液にそれぞれ５ｍｇ、１０ｍｇ、２０ｍｇを溶かし、フイルムを作製した。その結果、Ｓ１ヌクレア−ゼを作用させなかったものは、１０ｍｇでもフイルム調製が困難であったが、Ｓ１ヌクレア−ゼを作用させ、二本鎖率を高めることによって、５ｍｇでもフイルム化できることが確認できた。また、高分子ＤＮＡにより作製したフイルムと比較すると、少し厚みのあるフイルムであった。このようにして作製された二本鎖３００−５００塩基対ＤＮＡフイルムが高分子フイルムより優れている点は、高分子ＤＮＡを用いた場合溶解することが困難な濃度である１００mg／ｍｌでも容易に溶解し、高分子ＤＮＡ（２５mg／ｍｌ）と比べると粘性がなく、扱い易いことである。 Films were compared using two types of S1 nuclease and those not. Films were prepared by dissolving 5 mg, 10 mg and 20 mg, respectively, in 200 μl of chloroform / ethanol solution. As a result, although it was difficult to prepare a film even when 10 mg was not allowed to act on S1 nuclease, it was confirmed that the film could be formed even at 5 mg by increasing the double-stranded rate by acting S1 nuclease. did it. Moreover, it was a film with a little thickness compared with the film produced with polymeric DNA. The double-stranded 300-500 base pair DNA film produced in this way is superior to the polymer film in that it can be easily dissolved even at a concentration of 100 mg / ml, which is difficult to dissolve when polymer DNA is used. It dissolves and is less viscous and easier to handle than high molecular DNA (25 mg / ml).
ＤＮＡは、π電子が豊富な塩基対がスタッキングし、かつ分子量が揃った不斉二重らせん構造をとる生体高分子であり、これらの構造に基づいた導電性、分子の配向化、インターカレーター吸着能といった特性を示す。これらの特性を生かした新機能素材の調製法が本発明である。本発明に係る分子量を調整した二本鎖ＤＮＡの収率が高い機能性素材としてのＤＮＡは、分子量が目的に合わせて調整してあることと、二本鎖ＤＮＡの収率が高いことによって、高機能性を発揮できる機能性素材となっており、例えばＤＮＡ有機導電体、非線型光学材料、ダイオキシン等の吸着剤、医療用素材等広い分野での利用が期待されている。 DNA is a biopolymer that has an asymmetric double helix structure in which base pairs rich in π electrons are stacked and molecular weights are uniform. Conductivity, molecular orientation, and intercalator adsorption are based on these structures. It shows characteristics such as performance. A method for preparing a new functional material that takes advantage of these characteristics is the present invention. The DNA as a functional material having a high yield of double-stranded DNA with an adjusted molecular weight according to the present invention has a molecular weight adjusted according to the purpose and a high yield of double-stranded DNA. It is a functional material that can exhibit high functionality, and is expected to be used in a wide range of fields such as DNA organic conductors, non-linear optical materials, adsorbents such as dioxins, and medical materials.
Claims (6)
Protease is added to high-purity polymer DNA as an enzyme capable of cleaving the DNA strand, and the enzyme treatment is carried out to adjust the cleaving to about 200-600 base pairs of DNA, and then single-stranded DNA can be cleaved specifically. By adding S1 nuclease as an enzyme and cleaving the single-stranded DNA by enzymatic treatment, and removing the cleaved single-stranded DNA, the proportion of double-stranded DNA is aligned to 300-500 base pairs. After preparing the DNA as a functional material that is high, the DNA solution and the lipid solution as a functional material that have the same 300-500 base pairs and a high proportion of double-stranded DNA are mixed and stirred to prepare DNA- A lipid complex was obtained, and the DNA-lipid complex was dissolved in a heat-bonded or organic solvent, which was then stretched into a thin film and dried to obtain a DNA film. Process for the preparation of highly functional DNA film characterized by and.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2004094775A JP4235817B2 (en) | 2004-03-29 | 2004-03-29 | A method for preparing DNA as a functional material and a method for preparing a highly functional DNA film using DNA as a functional material. |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2004094775A JP4235817B2 (en) | 2004-03-29 | 2004-03-29 | A method for preparing DNA as a functional material and a method for preparing a highly functional DNA film using DNA as a functional material. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2005278444A true JP2005278444A (en) | 2005-10-13 |
| JP4235817B2 JP4235817B2 (en) | 2009-03-11 |
Family
ID=35177503
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2004094775A Expired - Fee Related JP4235817B2 (en) | 2004-03-29 | 2004-03-29 | A method for preparing DNA as a functional material and a method for preparing a highly functional DNA film using DNA as a functional material. |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4235817B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101713119B1 (en) * | 2016-04-15 | 2017-03-08 | 한국기계연구원 | Manufacturing method of salmon deoxyribonucleic acid film and apparatus for injecting medicine using the salmon deoxyribonucleic acid film |
| WO2017179956A3 (en) * | 2016-04-15 | 2017-12-07 | 한국기계연구원 | Method for manufacturing nucleic acid film and apparatus for injecting medicine using nucleic acid film |
-
2004
- 2004-03-29 JP JP2004094775A patent/JP4235817B2/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101713119B1 (en) * | 2016-04-15 | 2017-03-08 | 한국기계연구원 | Manufacturing method of salmon deoxyribonucleic acid film and apparatus for injecting medicine using the salmon deoxyribonucleic acid film |
| WO2017179956A3 (en) * | 2016-04-15 | 2017-12-07 | 한국기계연구원 | Method for manufacturing nucleic acid film and apparatus for injecting medicine using nucleic acid film |
| JP2019519472A (en) * | 2016-04-15 | 2019-07-11 | エーディーエムバイオサイエンス インコーポレイテッド | Method for producing nucleic acid film and drug injection apparatus using nucleic acid film |
| US11040103B2 (en) | 2016-04-15 | 2021-06-22 | Admbioscience Inc. | Method for manufacturing nucleic acid film and apparatus for injecting medicine using nucleic acid film |
| JP2022008985A (en) * | 2016-04-15 | 2022-01-14 | エーディーエムバイオサイエンス インコーポレイテッド | Manufacturing method of nucleic acid film and medicine injection device using nucleic acid film |
| JP7034425B2 (en) | 2016-04-15 | 2022-03-14 | エーディーエムバイオサイエンス インコーポレイテッド | Nucleic acid film manufacturing method and drug injection device using nucleic acid film |
| JP7191346B2 (en) | 2016-04-15 | 2022-12-19 | エーディーエムバイオサイエンス インコーポレイテッド | Manufacturing method of nucleic acid film and drug injection device using nucleic acid film |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4235817B2 (en) | 2009-03-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mout et al. | Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing | |
| Kick et al. | Efficient production of single-stranded phage DNA as scaffolds for DNA origami | |
| Mbous et al. | Applications of deep eutectic solvents in biotechnology and bioengineering—Promises and challenges | |
| Lyu et al. | Liposome crosslinked polyacrylamide/DNA hydrogel: a smart controlled‐release system for small molecular payloads | |
| Xu et al. | Combined cross-linked enzyme aggregates as biocatalysts | |
| Carneiro et al. | Long-range assembly of DNA into nanofibers and highly ordered networks using a block copolymer approach | |
| Gil et al. | Click chemistry-what’s in a name? Triazole synthesis and beyond | |
| Xu et al. | Hydrophobic-region-induced transitions in self-assembled peptide nanostructures | |
| Osada et al. | Engineering RNA–protein complexes with different shapes for imaging and therapeutic applications | |
| Hol et al. | The α-helix dipole and the properties of proteins | |
| Estroff | Introduction: biomineralization | |
| Xu et al. | Twisted nanotubes formed from ultrashort amphiphilic peptide I3K and their templating for the fabrication of silica nanotubes | |
| Ning et al. | Occlusion of sulfate-based diblock copolymer nanoparticles within calcite: effect of varying the surface density of anionic stabilizer chains | |
| Izumrudov et al. | Interpolyelectrolyte complexes: Advances and prospects of application | |
| Yang et al. | Shear-thinning and designable responsive supramolecular DNA hydrogels based on chemically branched DNA | |
| Kim et al. | Effect of protein surface charge distribution on protein–polyelectrolyte complexation | |
| Velema et al. | Fluorogenic templated reaction cascades for RNA detection | |
| Lam et al. | The effect of protein electrostatic interactions on globular protein–polymer block copolymer self-assembly | |
| Xia et al. | Poly (L-lysine)-mediated biomimetic silica synthesis: effects of mixing sequences and counterion concentrations | |
| KR100700483B1 (en) | Rapid dehydration of proteins | |
| Cho et al. | Improved glucose‐neopentyl glycol (GNG) amphiphiles for membrane protein solubilization and stabilization | |
| Iacomino et al. | DNA is wrapped by the nuclear aggregates of polyamines: the imaging evidence | |
| WO2023114090A2 (en) | Signal boost cascade assay | |
| Hazam et al. | Topological effects on the designability and bactericidal potency of antimicrobial peptides | |
| Köse | Nucleotide incorporated magnetic microparticles for isolation of DNA |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20080513 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20080710 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20080812 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20081009 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20081104 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20081202 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 4235817 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111226 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111226 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121226 Year of fee payment: 4 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121226 Year of fee payment: 4 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131226 Year of fee payment: 5 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313111 |
|
| R360 | Written notification for declining of transfer of rights |
Free format text: JAPANESE INTERMEDIATE CODE: R360 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |