JP3996103B2 - Nucleic acid treatment agent and nucleic acid treatment method - Google Patents

Nucleic acid treatment agent and nucleic acid treatment method Download PDF

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JP3996103B2
JP3996103B2 JP2003273264A JP2003273264A JP3996103B2 JP 3996103 B2 JP3996103 B2 JP 3996103B2 JP 2003273264 A JP2003273264 A JP 2003273264A JP 2003273264 A JP2003273264 A JP 2003273264A JP 3996103 B2 JP3996103 B2 JP 3996103B2
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静昭 村田
寧 陳
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National Institute of Japan Science and Technology Agency
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本発明は、核酸処理剤及び核酸の処理方法に関する。更に詳しくは、本発明は、蛍光特性部であるプテリジン構造体と核酸凝縮化機能部であるポリアミンとを備え、核酸の凝縮可視化剤として用いられる核酸処理剤と、核酸処理剤を用いて核酸の凝縮可視化を行い、更には in vitro 又は in vivoでの核酸の遺伝子操作又は遺伝子機能発現のために、凝縮可視化した核酸の凝縮状態の解除を、二重らせん構造の解裂が可能な状態で行う核酸の処理方法に関する。   The present invention relates to a nucleic acid treatment agent and a nucleic acid treatment method. More specifically, the present invention comprises a pteridine structure that is a fluorescence characteristic part and a polyamine that is a nucleic acid condensation function part, and a nucleic acid treatment agent used as a nucleic acid condensation visualization agent, and a nucleic acid treatment agent using the nucleic acid treatment agent. Condensation visualization is performed, and further, the condensed state of the nucleic acid visualized by condensation is released in a state where the double helix structure can be cleaved for genetic manipulation or gene function expression of the nucleic acid in vitro or in vivo. The present invention relates to a method for treating nucleic acid.

核酸、特にＤＮＡの凝縮（折り畳み）現象を制御することは、例えば、遺伝子治療においてリポソームからなる人工ベクターに遺伝子を封入し、これを標的細胞内に送り込むためには必須のテクニックである。その際、核酸を蛍光色素等で染色し可視化することは、顕微鏡下での取り扱いを容易にする。   Controlling the condensation (folding) phenomenon of nucleic acids, particularly DNA, is an indispensable technique for encapsulating a gene in an artificial vector composed of a liposome in gene therapy, for example, and feeding it into a target cell. At that time, staining and visualizing the nucleic acid with a fluorescent dye or the like facilitates handling under a microscope.

従来、核酸凝縮化剤としてはポリアミン等の多価カチオンが公知であり、一方、核酸を可視化するための蛍光色素としてＤＡＰＩ（４’，６−ジアミジノ−２−フェニルインドールジヒドロクロライド）等が公知である。   Conventionally, polyvalent cations such as polyamines are known as nucleic acid condensing agents, while DAPI (4 ′, 6-diamidino-2-phenylindole dihydrochloride) is known as a fluorescent dye for visualizing nucleic acids. is there.

"Discrete Coil-Grobule Transition of Single Duplex DNAs Induced by Polyamines", M. Takahashi, K. Yoshikawa, V. V. Vasilevskaya, and A. R. Khokhlov, J. Phys. Chem. B, 101, 9396-9401, (1997)"Discrete Coil-Grobule Transition of Single Duplex DNAs Induced by Polyamines", M. Takahashi, K. Yoshikawa, V. V. Vasilevskaya, and A. R. Khokhlov, J. Phys. Chem. B, 101, 9396-9401, (1997) "Direct Visualization of Individual DNA Moleculesby Fluorescence Microscopy; Characterization of the Factors AffectingSignal/Background and Optimization of Imazing Conditions Using YOYO",Sergio Gurrieri, K. Sam Wells, Iain D. Johnson, and Carlos Bustamante, Analytical biochemistry, 249, 44-53, (1997) 上記の非特許文献１には、ＤＮＡを凝縮化させる凝縮化剤として、ポリアミンを用いることが開示されている。上記の非特許文献２には、ＤＮＡを可視化させる蛍光色素として、ＹＯＹＯを用いることが開示されている。「ＹＯＹＯ」とは、「１，１’−（４，４，７，７−テトラメチル−４，７−ジアザウンデカメチレン）−ビス−４−〔３−メチル−２，３−ジヒドロ−（ベンゾ−１，３−オキサゾル）−２−メチリデン〕−キノリニウムテトライオダイド」を言い、当業者に周知の化合物である。"Direct Visualization of Individual DNA Moleculesby Fluorescence Microscopy; Characterization of the Factors AffectingSignal / Background and Optimization of Imazing Conditions Using YOYO", Sergio Gurrieri, K. Sam Wells, Iain D. Johnson, and Carlos Bustamante, Analytical biochemistry, 249, 44- 53, (1997) The above Non-Patent Document 1 discloses the use of polyamine as a condensing agent for condensing DNA. Non-Patent Document 2 discloses using YOYO as a fluorescent dye for visualizing DNA. “YOYO” means “1,1 ′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene) -bis-4- [3-methyl-2,3-dihydro-”. (Benzo-1,3-oxazol) -2-methylidene] -quinolinium tetraiodide ", a compound well known to those skilled in the art.

ところで、従来は、核酸を凝縮化させ可視化して取り扱うことを考えた場合、核酸凝縮化剤による核酸の凝縮化操作と蛍光色素等による染色操作とをそれぞれ行う必要があるため、面倒であった。   By the way, conventionally, when it was considered to condense and visualize nucleic acid, it was troublesome because it was necessary to perform a condensation operation of the nucleic acid with a nucleic acid condensing agent and a staining operation with a fluorescent dye, respectively. .

又、技術的な目的からすると、凝縮化された核酸だけを蛍光色素等により可視化したい訳であるが、それぞれ別の試薬によって別のプロセスとして行う凝縮化と染色とを良好に同調させ得るか否かと言う不安があった。核酸の凝縮化と染色が良好に同調しない場合、例えば凝縮化された核酸と十分に凝縮化されていない核酸とを蛍光強度によって明瞭に識別できないと言う不具合や、凝縮化された核酸による蛍光をバックグラウンドからの蛍光と明瞭に識別できないと言う不具合があった。   Also, for technical purposes, we would like to visualize only the condensed nucleic acid with a fluorescent dye, etc., but it is possible to satisfactorily synchronize the condensation and staining performed as separate processes with different reagents. I was worried. If the condensation and staining of the nucleic acid do not synchronize well, for example, there is a problem that the condensed nucleic acid and the nucleic acid that is not sufficiently condensed cannot be clearly distinguished by the fluorescence intensity, or the fluorescence caused by the condensed nucleic acid. There was a problem that it could not be clearly distinguished from the fluorescence from the background.

次に、従来の核酸凝縮化剤には、例えば発ガン性を示す等と言う、細胞又は生物体に対して有害なものもあった。一方、細胞や生物体に対して無毒性であると考えられる一定のポリアミン等の核酸凝縮化剤は、その核酸凝縮化機能が必ずしも十分に強くなかった。   Next, some conventional nucleic acid condensing agents are harmful to cells or organisms, such as exhibiting carcinogenicity. On the other hand, certain nucleic acid condensing agents such as polyamines considered to be non-toxic to cells and organisms did not necessarily have a sufficiently strong nucleic acid condensing function.

更に、遺伝子たる核酸を凝縮可視化する操作では、その後に核酸の凝縮化状態を解除して、 in vitro で各種の遺伝子操作を行ったり、 in vivoで遺伝子機能を発現させたりしたい場合が多い。このような場合、凝縮化した核酸に対して、人為的操作により又は核酸の細胞内への導入によって希釈化やイオン強度（塩濃度）の変化をもたらすと、凝縮化状態が解除されることが了知されている。   Furthermore, in the operation of condensing and visualizing the nucleic acid that is a gene, it is often desirable to subsequently release the condensed state of the nucleic acid and perform various gene manipulations in vitro or to express gene functions in vivo. In such a case, if the condensed nucleic acid is diluted or changed in ionic strength (salt concentration) by artificial manipulation or introduction of the nucleic acid into a cell, the condensed state may be released. It is known.

しかしながら、核酸の凝縮化状態を解除した際、核酸可視化剤が核酸から脱離するかどうかは別な問題である。核酸可視化剤には、核酸に対して電気化学的に結合するタイプと、核酸の二重らせん構造にインターカレートされるタイプとがある。インターカレータタイプの核酸可視化剤は、核酸の凝縮化状態を解除しても、核酸から脱離しない。その結果、核酸の二重らせん構造は、インターカレートされた可視化剤によって開裂が阻害されるため、ＰＣＲ法による遺伝子の増幅、細胞内での遺伝子融合又は遺伝子組替え、あるいは in vitro 又は in vivoでの遺伝子の転写又は複製等に供することができなかった。   However, whether the nucleic acid visualization agent is desorbed from the nucleic acid when the condensed state of the nucleic acid is released is another problem. Nucleic acid visualization agents include a type that binds electrochemically to a nucleic acid and a type that intercalates into the double helix structure of a nucleic acid. The intercalator type nucleic acid visualization agent does not desorb from the nucleic acid even when the condensed state of the nucleic acid is released. As a result, the double helix structure of the nucleic acid is inhibited from being cleaved by the intercalated visualization agent. Therefore, gene amplification by PCR, gene fusion or recombination in cells, or in vitro or in vivo The gene could not be used for transcription or replication of the gene.

従来技術においては、このような不具合を回避するために、核酸の凝縮可視化操作と、核酸の凝縮化状態の解除を前提とする核酸の凝縮化操作とを、別々に切り離して行わねばならなかった。即ち、第１の実験として、核酸凝縮化剤と核酸可視化剤を併用した核酸の凝縮可視化操作を行い、これによって核酸凝縮化に必要な核酸凝縮化剤の種類、使用量、使用条件等のデータを得る。次に、第２の実験として、これらのデータを参照しながら、核酸可視化剤を併用せずに、核酸の凝縮化状態の解除を前提とする核酸の凝縮化操作を行う。このような二段階の実験操作は非常に面倒であり、研究の効率を低下させ、かつ、いわば「目隠し」の状態で第２の実験を行うため、その信頼性を損ないかねないものであった。   In the prior art, in order to avoid such problems, it was necessary to separate the nucleic acid condensation visualization operation and the nucleic acid condensation operation on the premise of releasing the nucleic acid condensation state separately. . That is, as a first experiment, a nucleic acid condensation visualization operation using both a nucleic acid condensing agent and a nucleic acid visualizing agent is performed, and thereby data such as the type, amount used, and usage conditions of the nucleic acid condensing agent necessary for nucleic acid condensation. Get. Next, as a second experiment, with reference to these data, a nucleic acid condensing operation is performed on the premise that the nucleic acid condensing state is released without using a nucleic acid visualization agent in combination. Such a two-stage experimental operation is very troublesome, which reduces the efficiency of the research and, in other words, performs the second experiment in a “blindfolded” state, so that its reliability may be impaired. .

そこで本発明は、従来技術における上記各種の不具合を解消することを、解決すべき技術的課題とする。   Therefore, the present invention has a technical problem to be solved to solve the above-described various problems in the prior art.

（第１発明の構成）
上記課題を解決するための本願第１発明の構成は、蛍光特性部であるプテリジン構造体の２位、４位及び６位の内の少なくとも一つの置換位置に、核酸凝縮化機能部であるポリアミンを導入した化学構造を有し、核酸の凝縮可視化剤として用いられる、核酸処理剤である。 (Configuration of the first invention)
The structure of the first invention of the present application for solving the above-described problem is that a polyamine which is a nucleic acid condensing function part is located at at least one substitution position among positions 2, 4 and 6 of a pteridine structure which is a fluorescent characteristic part. It is a nucleic acid treatment agent that has a chemical structure in which is introduced and is used as a nucleic acid condensation visualization agent.

（第２発明の構成）
上記課題を解決するための本願第２発明の構成は、
前記第１発明に係る核酸処理剤が次の（１）及び／又は（２）の特性を有する、核酸処理剤である。
（１）ポリアミン部分による核酸凝縮化機能が、当該ポリアミンを単独の化学構造体として用いた場合に比較して、強化されている。
（２）核酸処理剤により凝縮可視化した核酸の凝縮状態が解除された際に、この核酸処理剤が核酸から脱離する。 (Configuration of the second invention)
The configuration of the second invention of the present application for solving the above problem is as follows.
The nucleic acid treating agent according to the first invention is a nucleic acid treating agent having the following characteristics (1) and / or (2).
(1) The nucleic acid condensation function by the polyamine moiety is enhanced as compared with the case where the polyamine is used as a single chemical structure.
(2) When the condensed state of the nucleic acid visualized by condensation with the nucleic acid treatment agent is released, the nucleic acid treatment agent is detached from the nucleic acid.

（第３発明の構成）
上記課題を解決するための本願第３発明の構成は、前記第１発明又は第２発明に係るプテリジン構造体に対するポリアミンの導入位置が、２位又は６位のいずれか一つの置換位置である、核酸処理剤である。 (Configuration of the third invention)
The configuration of the third invention of the present application for solving the above problem is that the polyamine introduction position with respect to the pteridine structure according to the first invention or the second invention is any one of the substitution positions at the 2-position or the 6-position, It is a nucleic acid treatment agent.

（第４発明の構成）
上記課題を解決するための本願第４発明の構成は、前記第１発明〜第３発明のいずれかに係るポリアミンが、１，３−ジアミノプロパン、スペルミジン又はスペルミンである、核酸処理剤である。 (Configuration of the fourth invention)
The structure of the fourth invention of the present application for solving the above-mentioned problems is a nucleic acid treating agent in which the polyamine according to any of the first to third inventions is 1,3-diaminopropane, spermidine or spermine.

（第５発明の構成）
上記課題を解決するための本願第５発明の構成は、前記第１発明〜第４発明のいずれかに係る核酸がＤＮＡである、核酸処理剤である。 (Structure of the fifth invention)
The structure of the fifth invention of the present application for solving the above-mentioned problem is a nucleic acid treating agent in which the nucleic acid according to any one of the first to fourth inventions is DNA.

（第６発明の構成）
上記課題を解決するための本願第６発明の構成は、蛍光特性部と核酸凝縮化機能部とを有する核酸処理剤を核酸に作用させることにより、核酸の凝縮化と、凝縮化された核酸における蛍光特性部の高密度化に基づく核酸の可視化とを同調させた核酸の凝縮可視化を行う、核酸の処理方法である。 (Structure of the sixth invention)
The configuration of the sixth invention of the present application for solving the above-described problem is that a nucleic acid treating agent having a fluorescence characteristic part and a nucleic acid condensation function part is allowed to act on the nucleic acid, thereby condensing the nucleic acid and in the condensed nucleic acid. This is a nucleic acid processing method for performing condensation visualization of nucleic acids in synchronization with visualization of nucleic acids based on the densification of fluorescent characteristic portions.

第６発明において、「蛍光特性部と核酸凝縮化機能部とを有する核酸処理剤」とは、必ずしも上記の第１発明〜第５発明に係る核酸処理剤に限定されず、要するに核酸に対する蛍光染色機能を発揮する蛍光特性部と、核酸に対する凝縮化機能を発揮する核酸凝縮化機能部とを併せ備える単一の化合物であれば、使用可能である。   In the sixth invention, the “nucleic acid treatment agent having a fluorescence characteristic part and a nucleic acid condensation function part” is not necessarily limited to the nucleic acid treatment agent according to the first to fifth inventions described above, and in short, fluorescent staining for nucleic acids. A single compound having both a fluorescence characteristic part that exhibits a function and a nucleic acid condensation function part that exhibits a condensation function for nucleic acids can be used.

（第７発明の構成）
上記課題を解決するための本願第７発明の構成は、蛍光特性部と核酸凝縮化機能部とを有する核酸凝縮可視化剤であって、凝縮可視化した核酸の凝縮状態が解除された際に核酸から脱離すると言う特性を示す核酸処理剤を核酸に作用させることにより、次の（３）、（４）を行う、核酸の処理方法である。
（３）核酸を凝縮可視化する際には、核酸の凝縮化と、凝縮化された核酸における蛍光特性部の高密度化に基づく核酸の可視化とを同調させた核酸の凝縮可視化を行い、
（４） in vitro 又は in vivoでの核酸の遺伝子操作又は遺伝子機能発現のために核酸の凝縮状態を解除させる際には、前記遺伝子操作又は遺伝子機能発現に対する阻害因子であり得る蛍光特性部を核酸から脱離させる。 (Structure of the seventh invention)
The configuration of the seventh invention of the present application for solving the above-described problem is a nucleic acid condensation visualization agent having a fluorescence characteristic part and a nucleic acid condensation function part, and the nucleic acid is condensed when the condensed nucleic acid is released from the condensed state. This is a nucleic acid treatment method in which the following (3) and (4) are carried out by causing a nucleic acid treating agent exhibiting the property of desorption to act on nucleic acids.
(3) When condensing and visualizing the nucleic acid, the condensation visualization of the nucleic acid is performed in synchronization with the nucleic acid condensation and the visualization of the nucleic acid based on the densification of the fluorescent characteristic portion in the condensed nucleic acid.
(4) When releasing the condensed state of the nucleic acid for genetic manipulation or gene function expression of the nucleic acid in vitro or in vivo, the fluorescent characteristic portion that may be an inhibitor for the genetic manipulation or gene function expression is added to the nucleic acid. Detach from.

（第８発明の構成）
上記課題を解決するための本願第８発明の構成は、前記第７発明における遺伝子操作又は遺伝子機能発現が、ＰＣＲ法による遺伝子の増幅、細胞内での遺伝子融合又は遺伝子組替え、あるいは、 in vitro 又は in vivoでの遺伝子の転写又は複製である、核酸の処理方法である。 (Configuration of the eighth invention)
The structure of the eighth invention of the present application for solving the above-described problems is that the gene manipulation or gene function expression in the seventh invention is such that gene amplification by PCR method, gene fusion or gene recombination in cells, or in vitro or A nucleic acid processing method that is transcription or replication of a gene in vivo.

（第９発明の構成）
上記課題を解決するための本願第９発明の構成は、前記第６発明〜第８発明のいずれかに係る核酸処理剤が細胞及び生物体に対して無毒性なものである、核酸の処理方法である。 (Structure of the ninth invention)
The structure of the ninth invention of the present application for solving the above-described problem is a nucleic acid treatment method in which the nucleic acid treatment agent according to any of the sixth to eighth inventions is non-toxic to cells and organisms. It is.

（第１０発明の構成）
上記課題を解決するための本願第１０発明の構成は、前記第６発明〜第９発明のいずれかに係る核酸処理剤として第１発明〜第５発明のいずれかに係る核酸処理剤を用いる、核酸の処理方法である。 (Configuration of the tenth invention)
The structure of the tenth invention of the present application for solving the above problems uses the nucleic acid treatment agent according to any one of the first invention to the fifth invention as the nucleic acid treatment agent according to any of the sixth invention to the ninth invention. A method for treating nucleic acids.

（第１発明の効果）
第１発明の核酸処理剤は、プテリジン構造体の２位、４位及び６位の内の少なくとも一つの置換位置にポリアミンを導入した単一の化学構造体であり、核酸の凝縮可視化剤として用いられる。ここにおいて「プテリジン構造体」とは、プテリジン骨格からなる化合物又はその置換基誘導体、プテリジン構造を含む骨格からなる化合物（例えば、フラビン系化合物）又はその置換基誘導体を言う。又、「ポリアミン」とは、ジアミン以上の多価アミン化合物を言う。 (Effect of the first invention)
The nucleic acid treating agent of the first invention is a single chemical structure in which a polyamine is introduced into at least one substitution position among the 2-position, 4-position and 6-position of the pteridine structure, and is used as a nucleic acid condensation visualization agent. It is done. Here, the “pteridine structure” means a compound having a pteridine skeleton or a substituent derivative thereof, a compound having a skeleton containing a pteridine structure (for example, a flavin compound) or a substituent derivative thereof. “Polyamine” refers to a polyamine compound that is higher than diamine.

プテリジン構造体が蛍光特性を有することは公知であり、一定の化学構造を持つポリアミンも核酸凝縮化剤として公知である。しかし、この両者を一体的に結合させた単一の化学構造体が、そのプテリジン構造体部分において蛍光特性を良好に維持するか否か、あるいはそのポリアミン部分において良好な核酸凝縮化機能を維持するか否か、と言う点については、化学常識からしても容易に予測できない。   It is known that pteridine structures have fluorescent properties, and polyamines having a certain chemical structure are also known as nucleic acid condensing agents. However, a single chemical structure in which the two are integrally bonded maintains good fluorescence characteristics in the pteridine structure part, or maintains a good nucleic acid condensation function in the polyamine part. The point of whether or not can not be easily predicted even from chemical common sense.

実際、本願発明者が試作した多様なプテリジン−ポリアミン複合構造体において、蛍光特性と核酸凝縮化機能とを良好に発揮できない化合物例が非常に多かった。そして本願発明者は、試行錯誤の末に、プテリジン構造体の２位、４位及び６位の内の少なくとも一つの置換位置にポリアミンを導入したプテリジン−ポリアミン複合構造体においてのみ、蛍光特性と核酸凝縮化機能とが良好に維持されると言う事実を突き止めたのである。更に驚くべきことに、このようなプテリジン−ポリアミン複合構造体の幾つかのものは、後述するように、ポリアミン部分の核酸凝縮化機能が著しく強化されていた。   In fact, in various pteridine-polyamine composite structures prototyped by the inventors of the present application, there are very many examples of compounds that cannot exhibit the fluorescence characteristics and the nucleic acid condensation function well. Then, the inventors of the present application, after trial and error, have fluorescence characteristics and nucleic acids only in the pteridine-polyamine composite structure in which polyamine is introduced into at least one substitution position among positions 2, 4, and 6 of the pteridine structure. They found the fact that the condensation function is well maintained. Surprisingly, some of such pteridine-polyamine complex structures have a significantly enhanced nucleic acid condensation function of the polyamine moiety, as described below.

このような第１発明の核酸処理剤は、ポリアミン部分による核酸の凝縮化機能と、プテリジン構造体部分による蛍光染色機能とを併せ持つので、単一の作用薬として核酸の凝縮化と蛍光染色とを同時に行うことができ、前記した従来技術のような面倒がない。   Since the nucleic acid treating agent of the first invention has both the function of condensing nucleic acid by the polyamine moiety and the function of fluorescent staining by the pteridine structure part, the nucleic acid condensing and fluorescent staining can be performed as a single agent. It can be performed at the same time, and there is no trouble as in the prior art described above.

次に、核酸の凝縮化と、凝縮化された核酸における蛍光特性部の高密度化に基づく核酸の可視化とが必ず同調して発現するので、核酸の凝縮化が起こっていることの確認が極めて容易であり、又、例えば凝縮可視化された核酸をレーザートラッピング等の手法で目的の部位に移動させることも容易である。そして、凝縮化された核酸と十分に凝縮化されていない核酸とを蛍光強度によって明瞭に識別できないと言う不具合や、凝縮化された核酸による蛍光をバックグラウンドからの蛍光と明瞭に識別できないと言う不具合を伴わない。即ち、核酸の凝縮可視化操作を確実かつ容易に行うことができる。   Next, since the nucleic acid condensation and the visualization of the nucleic acid based on the high density of the fluorescence characteristic part in the condensed nucleic acid are always expressed in synchronism, it is extremely difficult to confirm that the nucleic acid is condensed. For example, it is easy to move the nucleic acid visualized by condensation, for example, to a target site by a technique such as laser trapping. In addition, it is said that the condensed nucleic acid and the nucleic acid that is not sufficiently condensed cannot be clearly distinguished by the fluorescence intensity, and the fluorescence caused by the condensed nucleic acid cannot be clearly distinguished from the fluorescence from the background. There are no defects. That is, the nucleic acid condensation visualization operation can be performed reliably and easily.

（第２発明の効果）
第２発明の核酸処理剤は、第１に、ポリアミン部分による核酸凝縮化機能が当該ポリアミンを単独の化学構造体として用いた場合に比較して強化されている。従って、より無害性で、より強力な核酸凝縮化剤の提供と言う世間の要求に応えることができるし、相対的に核酸凝縮化剤の使用量を低減させることができるので、その点に基づく種々のメリットも享受できる。 (Effect of the second invention)
In the nucleic acid treating agent of the second invention, firstly, the nucleic acid condensation function by the polyamine moiety is enhanced as compared with the case where the polyamine is used as a single chemical structure. Therefore, it is possible to meet the public demand for providing a more harmless and stronger nucleic acid condensing agent, and the amount of the nucleic acid condensing agent used can be relatively reduced. You can also enjoy various merits.

第２発明の核酸処理剤は、第２に、これにより凝縮可視化した核酸の凝縮状態が解除された際に、この核酸処理剤が核酸から脱離する。このような脱離特性が、蛍光特性部と核酸凝縮化機能部とを一体化させた化合物ならば必ず得られるのか、あるいは第１発明の核酸処理剤のようにプテリジン構造体にポリアミンを導入した化合物であるために得られたのかは、未だ明確ではない。   Secondly, the nucleic acid treatment agent of the second invention is detached from the nucleic acid when the condensed state of the nucleic acid condensed and visualized thereby is released. If such a desorption characteristic is a compound in which a fluorescence characteristic part and a nucleic acid condensation function part are integrated, a polyamine is introduced into the pteridine structure as in the nucleic acid treatment agent of the first invention. Whether it was obtained because it is a compound is not yet clear.

上記のような脱離特性を持つ核酸処理剤を使用する場合には、核酸の凝縮可視化操作の後、核酸の凝縮状態を解除させる際には、同時に核酸処理剤が核酸から脱離するので、非常に有利である。即ち、核酸の凝縮状態の解除と同時に蛍光特性部が核酸から脱離するので、核酸の二重らせん構造の開裂が支障なく起こり、その後の in vitro 又は in vivoでの核酸の遺伝子操作又は遺伝子機能発現等を問題なく行わせることができる。   When using a nucleic acid treatment agent having the above-described desorption properties, the nucleic acid treatment agent is desorbed from the nucleic acid at the same time when the nucleic acid condensation state is released after the nucleic acid condensation visualization operation. Very advantageous. That is, the fluorescence characteristic part is detached from the nucleic acid simultaneously with the release of the condensed state of the nucleic acid, so that the cleavage of the double helix structure of the nucleic acid occurs without trouble, and the subsequent genetic manipulation or gene function of the nucleic acid in vitro or in vivo. Expression and the like can be performed without problems.

このため、前記したような、核酸の凝縮可視化操作と、核酸の凝縮化状態の解除を前提とする核酸の凝縮化操作とを別々に切り離して二段階で行うと言う研究者の苦労が解消される。又、核酸可視化剤を併用せずに核酸凝縮化剤のみを用いると言う「目隠し」状態の核酸凝縮化操作による、実験の信頼性に対する不安からも開放される。   This eliminates the labor of researchers to separate the nucleic acid condensation visualization operation and the nucleic acid condensation operation on the premise of releasing the condensed nucleic acid state in two steps as described above. The Further, it is freed from anxiety about the reliability of the experiment by the “blindfolded” nucleic acid condensation operation in which only the nucleic acid condensing agent is used without using the nucleic acid visualizing agent.

（第３発明の効果）
第３発明のように、プテリジン構造体に対するポリアミンの導入位置が、２位又は６位のいずれか一つの置換位置である場合には、当該ポリアミンを単独の化学構造体として用いた場合に比較して、ポリアミン部分の核酸凝縮化機能が特に著しく強化される。 (Effect of the third invention)
As in the third invention, when the polyamine is introduced into the pteridine structure at any one of the 2-position and 6-position, the polyamine is used as a single chemical structure. Thus, the nucleic acid condensation function of the polyamine moiety is particularly significantly enhanced.

（第４発明の効果）
核酸処理剤の構成要素であるポリアミンの種類は限定されないが、第４発明のように、１，３−ジアミノプロパン、スペルミジン又はスペルミンを特に好ましく例示することができる。 (Effect of the fourth invention)
Although the kind of polyamine which is a component of the nucleic acid treating agent is not limited, 1,3-diaminopropane, spermidine or spermine can be particularly preferably exemplified as in the fourth invention.

（第５発明の効果）
核酸処理剤の処理対象である核酸の種類は特段限定されず、例えば、ＲＮＡや、ＲＮＡ−ＤＮＡハイブリッド鎖等も包含されるが、二重らせん構造を形成すると言う特性や、典型的な核酸凝縮化現象を発現すると言う点から、特にＤＮＡが好ましい。 (Effect of the fifth invention)
The type of nucleic acid to be processed by the nucleic acid treatment agent is not particularly limited, and includes, for example, RNA and RNA-DNA hybrid strands. DNA is particularly preferable from the viewpoint of expressing the oxidization phenomenon.

（第６発明の効果）
第６発明においては、蛍光特性部と核酸凝縮化機能部とを有する核酸処理剤を核酸に作用させるので、単一の作用薬により核酸の凝縮化と蛍光染色とを同時に行うことができ、研究効率が向上する。 (Effect of the sixth invention)
In the sixth invention, since the nucleic acid treatment agent having the fluorescence characteristic part and the nucleic acid condensation function part is allowed to act on the nucleic acid, it is possible to simultaneously perform the condensation and fluorescence staining of the nucleic acid with a single agent. Efficiency is improved.

又、核酸の凝縮化と、凝縮化された核酸における蛍光特性部の高密度化に基づく核酸の可視化とが必ず同調して発現するので、凝縮化された核酸の蛍光強度による識別を明瞭に行うことができ、核酸の凝縮可視化操作を確実かつ容易に行うことができる。   In addition, since the condensation of nucleic acids and the visualization of nucleic acids based on the high density of fluorescent characteristic portions in the condensed nucleic acids are always expressed in synchronism, distinction by the fluorescence intensity of the condensed nucleic acids is clearly performed. The nucleic acid condensation and visualization can be performed reliably and easily.

（第７発明の効果）
第７発明においては、上記の第６発明と同様の効果に加え、凝縮可視化操作による所定の実験目的を達した後、 in vitro 又は in vivoでの核酸の遺伝子操作又は遺伝子機能発現のために核酸の凝縮状態を解除させる際に、核酸処理剤（蛍光特性部）が核酸から脱離する。 (Effect of the seventh invention)
In the seventh invention, in addition to the same effects as in the sixth invention, the nucleic acid is used for in vitro or in vivo nucleic acid genetic manipulation or gene function expression after achieving a predetermined experimental purpose by condensation visualization. When the condensed state is released, the nucleic acid treatment agent (fluorescence characteristic part) is desorbed from the nucleic acid.

即ち、遺伝子操作又は遺伝子機能発現に対する阻害因子であり得る蛍光特性部が排除されるので、核酸の二重らせん構造の開裂が阻害されず、従ってその後の遺伝子操作又は遺伝子機能発現を支障なく行わせることができる。   That is, since the fluorescent characteristic portion that can be an inhibitor for gene manipulation or gene function expression is eliminated, cleavage of the double helix structure of the nucleic acid is not inhibited, so that subsequent gene manipulation or gene function expression can be performed without hindrance. be able to.

又、同じ目的の達成のために従来は必要であった、核酸の凝縮可視化操作と核酸の凝縮化状態の解除を前提とする核酸の凝縮化操作とを切り離して二段階で行うと言う面倒が不要となり、核酸可視化剤を併用せずに「目隠し」状態で行っていた核酸凝縮化操作を回避できる。   In addition, it is troublesome to separate the nucleic acid condensation visualization operation and the nucleic acid condensation operation, which are necessary in the past to release the condensed state of the nucleic acid, in two steps, which was conventionally necessary to achieve the same purpose. This eliminates the need for nucleic acid condensing operations performed in a “blindfolded” state without using a nucleic acid visualization agent.

（第８発明の効果）
上記の第７発明で言う「遺伝子操作又は遺伝子機能発現」の内容や種類は限定されないが、第８発明に規定するように、ＰＣＲ法による遺伝子の増幅、細胞内での遺伝子融合又は遺伝子組替え、あるいは、 in vitro 又は in vivoでの遺伝子の転写又は複製を好ましく例示することができる。 (Effect of the eighth invention)
The contents and types of “gene manipulation or gene function expression” referred to in the seventh invention are not limited, but as defined in the eighth invention, gene amplification by PCR method, gene fusion or gene recombination in cells, Alternatively, transcription or replication of genes in vitro or in vivo can be preferably exemplified.

（第９発明の効果）
核酸の処理方法に用いる核酸処理剤としては、細胞及び生物体に対して無毒性なものであることが、より好ましい。第１発明〜第５発明に係る核酸処理剤は、特殊な化学構造のポリアミンを含むものは別としても、一般的に細胞や生物体内に存在するプテリジン構造体及びポリアミンからなるので、少なくとも細胞及び生物体に対して無毒性である。 (Effect of the ninth invention)
The nucleic acid treatment agent used in the nucleic acid treatment method is more preferably non-toxic to cells and organisms. Since the nucleic acid treating agent according to the first to fifth inventions is composed of a pteridine structure and a polyamine that are generally present in cells and organisms, apart from those containing polyamines having a special chemical structure, at least the cells and Non-toxic to living organisms.

従って、核酸処理剤によって処理した核酸についての所定の操作目的を達成した後、そのままであるいは凝縮化状態を解除したもとで、更に in vivoの細胞実験等に供したり、又は医療目的から実験動物体内や人体内に導入する場合、これらの細胞及び生物体に対して有害な作用を起こさない。   Therefore, after achieving the predetermined operational purpose for the nucleic acid treated with the nucleic acid treating agent, it can be used as it is or after the condensed state is released, for further in vivo cell experiments, or for medical purposes. When introduced into the body or human body, it does not cause harmful effects on these cells and organisms.

（第１０発明の効果）
核酸の処理方法に用いる核酸処理剤としては、第１発明〜第５発明に係る核酸処理剤を特に好ましく例示することができる。 (Effect of the tenth invention)
As the nucleic acid treatment agent used in the nucleic acid treatment method, the nucleic acid treatment agents according to the first to fifth inventions can be particularly preferably exemplified.

次に、本願の第１発明〜第１０発明を実施するための形態を、その最良の形態を含めて説明する。以下において、単に「本発明」と言う時は、本願の各発明を一括して指している。   Next, modes for carrying out the first invention to the tenth invention of the present application will be described including the best mode. In the following, the term “present invention” refers to each invention of the present application collectively.

〔核酸処理剤〕
本発明の核酸処理剤は、核酸に対する蛍光染色機能を有する蛍光特性部としてのプテリジン構造体に対して、その２位、４位及び６位の内の少なくとも一つの置換位置に、核酸に対する凝縮化機能を有する核酸凝縮化機能部としてのポリアミンを導入した化学構造を有するものであり、核酸の凝縮可視化剤として用いられる。 [Nucleic acid treatment agent]
The nucleic acid treatment agent of the present invention condenses the nucleic acid at at least one of the 2-position, 4-position and 6-position with respect to the pteridine structure as a fluorescent characteristic part having a fluorescence staining function for the nucleic acid. It has a chemical structure in which polyamine is introduced as a functional nucleic acid condensation function part, and is used as a nucleic acid condensation visualization agent.

ポリアミンは、プテリジン構造体の２位、４位及び６位のいずれか一つの置換位置だけに導入されていても良いが、これらの二つ以上の置換位置に導入されていても良い。プテリジン構造体においてポリアミンが導入されていない任意の置換位置には、本発明の効果を阻害しない限りにおいて、任意の置換基を導入することができる。上記の「任意の置換位置」とは、２位、４位及び６位のうちポリアミンが導入されていない置換位置と、その他の置換位置とを含む。   The polyamine may be introduced only at one of the substitution positions of the 2-position, 4-position and 6-position of the pteridine structure, but may be introduced at these two or more substitution positions. In the pteridine structure, an arbitrary substituent can be introduced at any substitution position where no polyamine is introduced as long as the effect of the present invention is not inhibited. The “arbitrary substitution position” includes a substitution position where no polyamine is introduced among the 2-position, 4-position and 6-position, and other substitution positions.

これらの核酸処理剤における第１の特徴点は、プテリジンを単独の蛍光染色剤として用いる場合に比較して、プテリジン構造体の蛍光染色機能が遜色なく維持されていることである。核酸処理剤における第２の特徴点は、ポリアミンを単独の核酸凝縮化剤として用いる場合に比較して、核酸処理剤のポリアミン部分の核酸凝縮化機能がむしろ強化されていることである。プテリジン構造体に対するポリアミンの導入位置が、２位又は６位のいずれか一つの置換位置である場合には、その核酸凝縮化機能が著しく強化されている。核酸処理剤における第３の特徴点は、後述する代表的なポリアミンの実施形態において、構成要素たるプテリジン構造体及びポリアミンの特性から見て、核酸処理剤が細胞及び生物体に対して発ガン性その他の毒性を持たないことである。   The first characteristic point of these nucleic acid treatment agents is that the fluorescence staining function of the pteridine structure is maintained as compared with the case where pteridine is used as a single fluorescence staining agent. The second feature of the nucleic acid treating agent is that the nucleic acid condensing function of the polyamine portion of the nucleic acid treating agent is rather enhanced as compared with the case where polyamine is used as a single nucleic acid condensing agent. When the polyamine is introduced into the pteridine structure at any one of the 2-position and 6-position, the nucleic acid condensation function is remarkably enhanced. The third feature of the nucleic acid treatment agent is that, in typical polyamine embodiments described later, the nucleic acid treatment agent is carcinogenic to cells and organisms in view of the characteristics of the pteridine structure and polyamine as constituent elements. It has no other toxicity.

核酸処理剤における第４の、特に重大な特徴点は、これらの核酸処理剤によって凝縮可視化させた核酸の凝縮状態を適宜な手段（希釈化、溶液のイオン強度の変化等）によって解除した場合、核酸処理剤が核酸から脱離し、ひいてはプテリジン構造体が核酸から脱離する、と言う事実である。   The fourth particularly important feature of the nucleic acid treatment agent is that the condensed state of the nucleic acid condensed and visualized by these nucleic acid treatment agents is released by appropriate means (dilution, change in ionic strength of the solution, etc.) This is the fact that the nucleic acid treating agent is desorbed from the nucleic acid, and thus the pteridine structure is desorbed from the nucleic acid.

以上の第１〜第４の特徴点に基づく効果は、前記の「発明の効果」の欄で述べた通りである。   The effects based on the above first to fourth feature points are as described in the section “Effects of the Invention”.

プテリジン構造体としては、プテリジンが例示される。発明の目的を阻害しない限りにおいて、プテリジンの任意の置換位置に任意の置換基が導入された誘導体も例示される。フラビン系化合物で例示されるようなプテリジン構造を含む骨格からなる化合物や、発明の目的を阻害しない限りにおいてその任意の置換位置に任意の置換基が導入された誘導体も例示される。   An example of the pteridine structure is pteridine. As long as the object of the invention is not inhibited, derivatives in which an arbitrary substituent is introduced at an arbitrary substitution position of pteridine are also exemplified. Examples thereof include compounds composed of a skeleton containing a pteridine structure as exemplified by flavin compounds and derivatives in which an arbitrary substituent is introduced at an arbitrary substitution position as long as the object of the invention is not impaired.

ポリアミンとは、要するにジアミン以上の多価アミン化合物を意味する。従って、炭素骨格構造や炭素−窒素骨格構造その他の任意の骨格構造を持つ多価アミン（例えば、トリエチレンジアミン等）はいずれも包含される。特に好ましい例が、細胞又は生物体に対する無害性が既に確認されており、あるいはそのような無害性が確実に予測されるポリアミンである。とりわけ好ましい例が、比較的低分子量の炭素直鎖の両端にアミノ基を有するジアミンや、このようなジアミンが直列状に（リニアに）連結された炭素−窒素骨格構造を有するポリアミンである。   In short, polyamine means a polyvalent amine compound higher than diamine. Accordingly, any polyvalent amine having a carbon skeleton structure, a carbon-nitrogen skeleton structure, or any other skeleton structure (for example, triethylenediamine) is included. Particularly preferred examples are polyamines that have already been confirmed to be innocuous to cells or organisms, or in which such innocence is reliably predicted. Particularly preferred examples are diamines having amino groups at both ends of a carbon linear chain having a relatively low molecular weight, and polyamines having a carbon-nitrogen skeleton structure in which such diamines are connected in series (linearly).

ポリアミンの代表的な具体例として、１，３−ジアミノプロパン、スペルミジン又はスペルミンを挙げることができる。これらの内のいずれかのポリアミンを含む核酸処理剤は、強い核酸凝縮化機能を発揮し、しかも細胞又は生物体に対して無毒性である。   Typical examples of polyamines include 1,3-diaminopropane, spermidine or spermine. A nucleic acid treatment agent containing any of these polyamines exhibits a strong nucleic acid condensation function and is non-toxic to cells or organisms.

プテリジン構造体の２位の置換位置に１，３−ジアミノプロパンが導入された核酸処理剤の化学構造式を下記の「化１」に、プテリジン構造体の２位の置換位置にスペルミジンが導入された核酸処理剤の化学構造式を下記の「化２」に、プテリジン構造体の２位の置換位置にスペルミンが導入された核酸処理剤の化学構造式を下記の「化３」に、それぞれ示す。   The chemical structural formula of the nucleic acid treating agent in which 1,3-diaminopropane is introduced at the 2-position substitution position of the pteridine structure is shown in “Chemical Formula 1” below, and spermidine is introduced at the 2-position substitution position of the pteridine structure. The chemical structural formula of the nucleic acid treating agent is shown in “Chemical Formula 2” below, and the chemical structural formula of the nucleic acid treating agent in which spermine is introduced at the 2-position substitution position of the pteridine structure is shown in “Chemical Formula 3” below. .

又、上記の「化１」及び「化３」に示された核酸処理剤の合成スキームの一例を図１に示す。図１において、中間生成物及び最終生成物の化学構造式の下部には、その生成物の百分比（％）による収率を表記している。 An example of a synthesis scheme of the nucleic acid treating agent shown in the above “Chemical Formula 1” and “Chemical Formula 3” is shown in FIG. In FIG. 1, the yield by percentage (%) of the product is shown at the bottom of the chemical structural formula of the intermediate product and the final product.

次に、プテリジン構造体の６位の置換位置に１，３−ジアミノプロパンが導入された核酸処理剤の化学構造式を下記の「化４」に、プテリジン構造体の６位の置換位置にスペルミンが導入された核酸処理剤の化学構造式を下記の「化５」に、それぞれ示す。   Next, the chemical structural formula of the nucleic acid treating agent in which 1,3-diaminopropane is introduced at the 6-position substitution position of the pteridine structure is shown in the following “Chemical Formula 4”, and the spermine is substituted at the 6-position substitution position of the pteridine structure. The chemical structural formulas of the nucleic acid treating agents into which are introduced are shown in the following “chemical formula 5”.

更に、プテリジン構造体の４位の置換位置に１，３−ジアミノプロパンが導入された核酸処理剤の化学構造式を下記の「化６」に、プテリジン構造体の４位の置換位置にスペルミンが導入された核酸処理剤の化学構造式を下記の「化７」に、それぞれ示す。 Furthermore, the chemical structural formula of the nucleic acid treating agent in which 1,3-diaminopropane is introduced at the 4-position substitution position of the pteridine structure is shown in “Chemical 6” below, and spermine is present at the 4-position substitution position of the pteridine structure. The chemical structural formulas of the introduced nucleic acid treating agents are shown in the following “Chemical Formula 7”.

又、上記の「化６」及び「化７」に示された核酸処理剤の合成スキームの一例を図２に示す。図２において、中間生成物及び最終生成物の構造式の下部には、その生成物の百分比による収率を表記している。 FIG. 2 shows an example of a synthesis scheme of the nucleic acid treatment agent shown in the above “Chemical 6” and “Chemical 7”. In FIG. 2, the yield by percentage of the product is shown at the bottom of the structural formula of the intermediate product and the final product.

〔核酸の処理方法〕
本発明に係る核酸の処理方法は、要するに核酸に対して適用可能であり、好ましくはＤＮＡに対して適用されるが、その他のポリヌクレオチド、例えばＲＮＡやＲＮＡ−ＤＮＡハイブリッド鎖等も適用の対象であり得る。核酸処理の目的に「凝縮化」が含まれることから、余り短い核酸は、事実上、処理の対象とならない。具体的には、例えば１０，０００塩基（塩基対）程度以上の核酸が対象物として好ましい。 [Method of treating nucleic acid]
The nucleic acid processing method according to the present invention is basically applicable to nucleic acids and is preferably applied to DNA, but other polynucleotides such as RNA and RNA-DNA hybrid chains are also applicable. possible. Since “condensation” is included in the purpose of nucleic acid processing, nucleic acids that are too short are practically not subject to processing. Specifically, for example, a nucleic acid having about 10,000 bases (base pairs) or more is preferable as the target.

本発明の核酸の処理方法において、核酸処理剤としては、上記した各種の核酸処理剤を好ましく使用できるが、それ以外の蛍光特性部と核酸凝縮化機能部とを有効に併せ備える単一の化合物も使用できる。例えば、プテリジン構造体以外の蛍光特性部とポリアミンからなる核酸凝縮化機能部とを併せ備える単一の化合物、プテリジン構造体とポリアミン以外の核酸凝縮化機能部とを併せ備える単一の化合物、プテリジン構造体以外の蛍光特性部とポリアミン以外の核酸凝縮化機能部とを併せ備える単一の化合物、等を使用できる可能性がある。   In the nucleic acid treatment method of the present invention, the above-mentioned various nucleic acid treatment agents can be preferably used as the nucleic acid treatment agent, but a single compound that effectively combines the other fluorescent property part and the nucleic acid condensation function part. Can also be used. For example, a single compound having both a fluorescence characteristic part other than a pteridine structure and a nucleic acid condensation function part made of polyamine, a single compound having a pteridine structure and a nucleic acid condensation function part other than polyamine, pteridine There is a possibility that a single compound having a fluorescence characteristic part other than a structure and a nucleic acid condensation function part other than polyamine may be used.

（第１の核酸の処理方法）
本発明に係る第１の核酸の処理方法は、核酸の凝縮可視化方法である。即ち、蛍光特性部と核酸凝縮化機能部とを有する核酸処理剤を核酸に作用させることにより、核酸の凝縮化と、凝縮化された核酸における蛍光特性部の高密度化に基づく核酸の可視化とを同調させた核酸の凝縮可視化を行う、と言う内容である。 (First nucleic acid treatment method)
The first nucleic acid processing method according to the present invention is a method for visualizing condensation of nucleic acids. That is, by allowing a nucleic acid treatment agent having a fluorescence characteristic part and a nucleic acid condensation function part to act on the nucleic acid, the nucleic acid is condensed and the nucleic acid is visualized based on the densification of the fluorescence characteristic part in the condensed nucleic acid. It is said that the condensation visualization of the nucleic acid synchronized with is performed.

第１の核酸の処理方法においては、核酸の凝縮可視化を行う目的は限定されず、凝縮可視化を行った後に、更に後工程として何らかの操作又は実験を行うか否かも限定されない。即ち、単に凝縮可視化のみを目的とする場合も含まれる。   In the first nucleic acid processing method, the purpose of performing the visualization of condensation of the nucleic acid is not limited, and it is not limited whether or not some operation or experiment is further performed as a subsequent step after the visualization of the condensation. In other words, a case where only the condensation visualization is intended is included.

（第２の核酸の処理方法）
本発明に係る第２の核酸の処理方法は、核酸の凝縮可視化を行い、かつ、その後に核酸の凝縮状態を解除する方法である。即ち、この方法は、凝縮可視化した核酸の凝縮状態が解除された際に、核酸処理剤が核酸から脱離すると言う特性を利用して、次の第１工程と第２工程とを行う。 (Second nucleic acid treatment method)
The second nucleic acid processing method according to the present invention is a method for visualizing the condensation of nucleic acids and then releasing the condensed state of the nucleic acids. That is, this method performs the following first step and second step by utilizing the characteristic that the nucleic acid treatment agent is desorbed from the nucleic acid when the condensed and visualized nucleic acid is released from the condensed state.

第１工程：核酸を凝縮可視化する際に、核酸の凝縮化と、凝縮化された核酸における蛍光特性部の高密度化に基づく核酸の可視化とを同調させた核酸の凝縮可視化を行う。   First step: When condensing and visualizing nucleic acid, the condensing and visualizing of the nucleic acid is performed in synchronization with the condensing of the nucleic acid and the visualization of the nucleic acid based on the densification of the fluorescent characteristic portion in the condensed nucleic acid.

第２工程：第１工程において凝縮可視化された核酸について、 in vitro 又は in vivoでの核酸の遺伝子操作又は遺伝子機能発現のために、核酸の凝縮状態を解除させる。この工程では、上記のように、遺伝子操作又は遺伝子機能発現に対する阻害因子であり得る蛍光特性部が核酸から脱離する。第２工程における遺伝子操作又は遺伝子機能発現としては、少なくともＰＣＲ法による遺伝子の増幅、細胞内での遺伝子融合又は遺伝子組替え、あるいは in vitro 又は in vivoでの遺伝子の転写又は複製が含まれる。   Second step: The nucleic acid condensed and visualized in the first step is released from the condensed state of the nucleic acid for genetic manipulation or gene function expression of the nucleic acid in vitro or in vivo. In this step, as described above, the fluorescence characteristic portion that can be an inhibitor for gene manipulation or gene function expression is detached from the nucleic acid. The gene manipulation or gene function expression in the second step includes at least gene amplification by PCR, gene fusion or gene recombination in cells, or transcription or replication of a gene in vitro or in vivo.

（実施例１：核酸処理剤の蛍光特性の確認）
前記した「化１」及び「化３」に示す核酸処理剤の１０−６モル水溶液をそれぞれ調製し、JASCO FP-6600 スペクトルフルオロメーターを用いて蛍光スペクトルを測定したところ、波長３６０ｎｍの紫外光による励起に対して、「化１」に示す核酸処理剤は波長４４７ｎｍの蛍光（強度１２４）を発し、「化３」に示す核酸処理剤は波長４４８ｎｍの蛍光（強度１２７）を発することを確認した。 (Example 1: Confirmation of fluorescence characteristics of nucleic acid treating agent)
A 10-6 molar aqueous solution of the nucleic acid treating agent shown in the above “Chemical Formula 1” and “Chemical Formula 3” was prepared, and the fluorescence spectrum was measured using a JASCO FP-6600 spectrum fluorometer. It was confirmed that the nucleic acid treatment agent shown in “Chemical Formula 1” emits fluorescence (intensity 124) at a wavelength of 447 nm and the nucleic acid treatment agent shown in “Chemical Formula 3” emits fluorescence (intensity 127) at a wavelength of 448 nm in response to excitation. .

（実施例２：核酸の凝縮可視化）
本実施例を行う前提として、核酸の凝縮化状態についての３種類のスタンダードな状態を図３〜図５の蛍光顕微鏡観察像によって示す。図３は核酸が凝縮化していない「coil」状態であり、図５は核酸が十分に凝縮化した「globule 」状態である。これらに対して、図４に示す状態は、核酸の凝縮化部と非凝縮化部とが共存する「coexist 」状態である。これらのスタンダードを基準として、以下の実験における効果判定を行った。 (Example 2: Visualization of condensation of nucleic acid)
As a premise for carrying out this example, three kinds of standard states of nucleic acid condensation states are shown by the fluorescence microscope observation images of FIGS. FIG. 3 shows a “coil” state where the nucleic acid is not condensed, and FIG. 5 shows a “globule” state where the nucleic acid is sufficiently condensed. On the other hand, the state shown in FIG. 4 is a “coexist” state in which the condensed portion and the non-condensed portion of the nucleic acid coexist. Based on these standards, the effects in the following experiments were determined.

又、本実施例においては、試料用核酸として、約１６５．６ｋｂｐの非凝縮化状態にある２本鎖ＤＮＡ（T4DNA, Nippon Gene社）を用いた。そして、このＤＮＡと本発明に係る各種の核酸処理剤とを含む以下の組成液を調製した。   In this example, a double-stranded DNA (T4DNA, Nippon Gene) in a non-condensed state of about 165.6 kbp was used as the sample nucleic acid. And the following composition liquid containing this DNA and the various nucleic acid processing agent which concerns on this invention was prepared.

水 ４６０μＬ
０．１Ｍ トリス緩衝液 ５０μＬ
２−メルカプトエタノール ２０μＬ
１０μＭ ＤＮＡ溶液 １０μＬ
核酸処理剤溶液 １０μＬ（濃度は後述）
この組成液の調製に当たっては、上記の所定量の水、トリス緩衝液、２−メルカプトエタノール及び前記従来技術の項で述べたＤＡＰＩからなる混液を調製した。次に、上記のＤＮＡ溶液を加えて緩やかに攪拌した。その後、５〜１０分間静置してから上記の核酸処理剤溶液を加えて緩やかに攪拌した。 460 μL of water
0.1 M Tris buffer 50 μL
2-mercaptoethanol 20 μL
10 μM DNA solution 10 μL
Nucleic acid treating agent solution 10 μL (concentration is described later)
In preparing this composition solution, a mixed solution comprising the above-mentioned predetermined amount of water, Tris buffer, 2-mercaptoethanol and DAPI described in the above-mentioned section of the prior art was prepared. Next, the above DNA solution was added and gently stirred. Then, after leaving still for 5 to 10 minutes, said nucleic acid processing agent solution was added and it stirred gently.

こうして調製した組成液を更に５〜１０分間静置してから、この組成液を試料液として常法に従い観察用プレパラートを調製し、図８に示す構成の蛍光顕微鏡を用いて観察した。   The composition solution thus prepared was further allowed to stand for 5 to 10 minutes, and then an observation preparation was prepared according to a conventional method using this composition solution as a sample solution, and observed using a fluorescence microscope having the configuration shown in FIG.

又、実施例２に対する比較例として、このＤＮＡと、前記従来技術の項で述べた蛍光染色剤であるＹＯＹＯ（ Molecular Probes 社製）と、後述する公知の各種タイプの核酸凝縮化剤とを含む以下の組成液を同上の要領で調製し、かつ同上の要領で蛍光顕微鏡観察に供した。   Further, as a comparative example with respect to Example 2, this DNA, YOYO (manufactured by Molecular Probes), which is the fluorescent staining agent described in the above-mentioned section of the prior art, and various types of known nucleic acid condensing agents described later are included. The following composition liquids were prepared in the same manner as described above, and were subjected to fluorescence microscope observation in the same manner as described above.

水 ４００μＬ
０．１Ｍ トリス緩衝液 ５０μＬ
２−メルカプトエタノール ２０μＬ
２μＭ ＹＯＹＯ溶液 １０μＬ
１０μＭ ＤＮＡ溶液 １０μＬ
公知核酸凝縮化剤溶液 １０μＬ（濃度は後述）
表１に、比較例の公知核酸凝縮化剤溶液としてスペルミンをそれぞれ５〜４０μＭの範囲内で濃度を種々に変更して用いた場合（表中、「ｓｐｍ」と表記された欄）、実施例の核酸処理剤溶液として前記「化３」に示す化合物をそれぞれ５〜３０μＭの範囲内で濃度を種々に変更して用いた場合（表中、「Ａ−ｓｐｍ」と表記された欄）、及び、実施例の核酸処理剤溶液として前記「化７」に示す化合物をそれぞれ１０〜７５μＭの範囲内で濃度を種々に変更して用いた場合（表中、「Ｃ−ｓｐｍ」と表記された欄）について、蛍光顕微鏡観察による核酸の凝縮化効果の判定結果を示す。表１中、破線で「−−−」と表記した項目はデータを出していない（以下同様）。 400 μL of water
0.1 M Tris buffer 50 μL
2-mercaptoethanol 20 μL
2 μM YOYO solution 10 μL
10 μM DNA solution 10 μL
10 μL of known nucleic acid condensing agent solution (concentration will be described later)
In Table 1, when spermine is used as a known nucleic acid condensing agent solution of a comparative example in various concentrations within the range of 5 to 40 μM (columns labeled “spm” in the table), Examples When the compound shown in the above-mentioned “Chemical Formula 3” is used in various concentrations within the range of 5 to 30 μM as the nucleic acid treating agent solution (in the table, the column indicated as “A-spm”), and When the compound shown in the above-mentioned “Chemical Formula 7” was used as the nucleic acid treating agent solution in the examples in various concentrations within the range of 10 to 75 μM (in the table, the column labeled “C-spm”) ) Shows the determination result of the condensation effect of the nucleic acid by observation with a fluorescence microscope. In Table 1, data indicated as “---” by a broken line does not output data (the same applies hereinafter).

表２に、比較例の公知核酸凝縮化剤溶液としてスペルミジンをそれぞれ１０μＭ〜１ｍＭの範囲内で濃度を種々に変更して用いた場合（表中、「ｓｐｄ」と表記された欄）、及び、実施例の核酸処理剤溶液として前記「化２」に示す化合物をそれぞれ０．２〜１００μＭの範囲内で濃度を種々に変更して用いた場合（表中、「Ａ−ｓｐｄ」と表記された欄）について、蛍光顕微鏡観察による核酸の凝縮化効果の判定結果を示す。 In Table 2, when spermidine is used as a known nucleic acid condensing agent solution of a comparative example in various concentrations within the range of 10 μM to 1 mM, respectively (in the table, the column labeled “spd”), and When the compound shown in the above-mentioned “Chemical Formula 2” was used in the Examples in various concentrations within the range of 0.2 to 100 μM (indicated as “A-spd” in the table). Column) shows the determination result of the condensation effect of nucleic acid by fluorescence microscope observation.

表３に、比較例の公知核酸凝縮化剤溶液として１，３−ジアミノプロパンをそれぞれ１〜１５０ｍＭの範囲内で濃度を種々に変更して用いた場合（表中、「ｄｉａ」と表記された欄）、実施例の核酸処理剤溶液として前記「化１」に示す化合物をそれぞれ２０μＭ〜２ｍＭの範囲内で濃度を種々に変更して用いた場合（表中、「Ａ−ｄｉａ」と表記された欄）、及び、実施例の核酸処理剤溶液として前記「化６」に示す化合物をそれぞれ２０μＭ〜４ｍＭの範囲内で濃度を種々に変更して用いた場合（表中、「Ｃ−ｄｉａ」と表記された欄）について、蛍光顕微鏡観察による核酸の凝縮化効果の判定結果を示す。 In Table 3, when 1,3-diaminopropane was used as a known nucleic acid condensing agent solution of a comparative example in various concentrations within the range of 1 to 150 mM (indicated in the table as “dia”) Column), when the compound shown in the above “Chemical Formula 1” is used in various concentrations within the range of 20 μM to 2 mM as the nucleic acid treating agent solution in the examples (in the table, it is expressed as “A-dia”). Column) and when the compound shown in the above-mentioned “Chemical Formula 6” is used in various concentrations within the range of 20 μM to 4 mM as the nucleic acid treating agent solution in the examples (“C-dia” in the table). Column)), the determination result of the condensation effect of nucleic acid by fluorescence microscope observation is shown.

（実施例２の評価）
表１〜表３より明らかなように、本発明に係る各種の核酸処理剤は、核酸に対する凝縮可視化効果が明瞭に認められた。 (Evaluation of Example 2)
As is clear from Tables 1 to 3, various nucleic acid treatment agents according to the present invention clearly recognized the condensation visualization effect on nucleic acids.

次に、本発明に係る各種の核酸処理剤を用いた実施例と、核酸処理剤におけるポリアミン部分に相当するポリアミン化合物と核酸可視化剤であるＹＯＹＯとをそれぞれ別個に用いた比較例との対比において、次の２点が確認された。   Next, in comparison between an example using various nucleic acid treatment agents according to the present invention and a comparative example using a polyamine compound corresponding to the polyamine moiety in the nucleic acid treatment agent and YOYO as a nucleic acid visualization agent, respectively. The following two points were confirmed.

第１に、実施例及び比較例に係る多数の蛍光顕微鏡観察像の提示は差し控えるが、可視化効果において、各実施例は比較例に対して少なくとも遜色がなかった。比較例の幾つかの場合において、蛍光顕微鏡観察像が鮮明さに欠け、「coil」、「coexist 」又は「globule 」の判定に苦労するものがあった。   First, the presentation of a large number of fluorescence microscope observation images according to the examples and comparative examples is refrained, but in the visualization effect, each example is at least inferior to the comparative example. In some cases of the comparative examples, the images observed with the fluorescence microscope were not clear, and there were some which were difficult to judge “coil”, “coexist” or “globule”.

第２に、各実施例は比較例に対して、核酸凝縮化効果が強化されていた。核酸凝縮化効果の強化は、プテリジン構造体に対するポリアミンの導入位置が２位又は６位の置換位置である核酸処理剤において、特に著しかった。   Secondly, the nucleic acid condensation effect was enhanced in each example compared to the comparative example. The enhancement of the nucleic acid condensation effect was particularly remarkable in the nucleic acid treatment agent in which the polyamine was introduced into the pteridine structure at the 2-position or 6-position.

（実施例３：核酸の凝縮状態の解除）
最初に、実施例２と同じ要領で、以下の第１の組成液を調製し、かつ、ＤＮＡの凝縮化の様子を蛍光顕微鏡で観察した。ＤＮＡは、Ｔ４ ベクターと連結された約１６５．６ｋｂｐの非凝縮化状態にある２本鎖ＤＮＡであり、「核酸処理剤溶液」とは前記の「化３」に示す核酸処理剤の溶液である。 (Example 3: Release of the condensed state of nucleic acid)
First, the following first composition solution was prepared in the same manner as in Example 2, and the state of DNA condensation was observed with a fluorescence microscope. The DNA is a double-stranded DNA in a non-condensed state of about 165.6 kbp linked to a T4 vector, and the “nucleic acid treating agent solution” is a solution of the nucleic acid treating agent shown in the above “Chemical Formula 3”. .

水 ４００μＬ
０．１Ｍ トリス緩衝液 ５０μＬ
２−メルカプトエタノール ２０μＬ
１０μＭ ＤＮＡ溶液 １０μＬ
２μＭ ＹＯＹＯ溶液 １０μＬ
１ｍＭ 核酸処理剤溶液 １０μＬ
上記の蛍光顕微鏡観察像を図６に示すが、十分な凝縮化状態にあるＤＮＡを、明瞭な蛍光によって識別・確認することができた。 400 μL of water
0.1 M Tris buffer 50 μL
2-mercaptoethanol 20 μL
10 μM DNA solution 10 μL
2 μM YOYO solution 10 μL
1 mM Nucleic Acid Treatment Solution 10 μL
The above fluorescence microscope observation image is shown in FIG. 6, and DNA in a sufficiently condensed state could be identified and confirmed by clear fluorescence.

次に、上記の組成液を１２，０００r.p.m.での５分間の遠心分離に供し、核酸処理剤により凝縮化されたＤＮＡを沈殿させてから上澄み液を捨て、この沈殿に対して下記の第２の組成液を注加した。   Next, the above composition solution is subjected to centrifugation at 12,000 rpm for 5 minutes to precipitate DNA condensed by the nucleic acid treating agent, and then the supernatant is discarded. 2 composition liquids were added.

水 ４００μＬ
０．１Ｍ トリス緩衝液 ５０μＬ
２−メルカプトエタノール ２０μＬ
２μＭ ＹＯＹＯ溶液 １０μＬ
そして容器を激しく振とうし、１０分間静置した後に、組成液を同上の要領で蛍光顕微鏡観察に供した。図７に示すその観察像によれば、ＤＮＡの５０％以上が凝縮化状態を解除されており、前記の凝縮化効果の判定基準によれば、典型的な「coexist 」状態であった。 400 μL of water
0.1 M Tris buffer 50 μL
2-mercaptoethanol 20 μL
2 μM YOYO solution 10 μL
The container was shaken vigorously and allowed to stand for 10 minutes, and then the composition solution was subjected to fluorescence microscope observation in the same manner as above. According to the observed image shown in FIG. 7, 50% or more of the DNA has been released from the condensed state, and according to the criteria for determining the condensation effect, a typical “coexist” state was obtained.

（実施例３の評価）
図６及び図７に示す結果より、次の点が確認された。即ち、核酸処理剤により凝縮化されたＤＮＡの沈殿に対する第２の組成液の注加によって、核酸処理剤が低濃度化されてＤＮＡから脱離し、ＤＮＡが凝縮化状態を解除された。 (Evaluation of Example 3)
The following points were confirmed from the results shown in FIGS. That is, by adding the second composition solution to the precipitate of DNA condensed by the nucleic acid treatment agent, the concentration of the nucleic acid treatment agent was reduced and desorbed from the DNA, and the condensed state of the DNA was released.

本発明によって、 in vitro 又は in vivoでの各種の遺伝子操作又は遺伝子機能発現、例えば遺伝子治療においてリポソームからなる人工ベクターに遺伝子を封入し、これを標的細胞内に送り込む等の操作に必要・有益な核酸の凝縮（折り畳み）現象の制御を、可視化したもとで自由に行うことが可能になる。   According to the present invention, various gene manipulations or gene function expressions in vitro or in vivo are necessary and beneficial for manipulations such as encapsulating a gene in an artificial vector composed of liposomes in gene therapy and feeding it into a target cell. It is possible to freely control the condensation (folding) phenomenon of nucleic acids under visualization.

核酸処理剤の合成スキームを示す図である。It is a figure which shows the synthetic scheme of a nucleic acid processing agent.

核酸処理剤の合成スキームを示す図である。It is a figure which shows the synthetic scheme of a nucleic acid processing agent.

核酸の非凝縮化状態を示す蛍光顕微鏡観察像である。It is a fluorescence-microscope observation image which shows the non-condensed state of a nucleic acid.

核酸の凝縮化部と非凝縮化部の共存状態を示す蛍光顕微鏡観察像である。It is a fluorescence microscope observation image which shows the coexistence state of the condensation part and non-condensation part of a nucleic acid.

核酸の十分な凝縮化状態を示す蛍光顕微鏡観察像である。It is a fluorescence-microscope observation image which shows the sufficient condensation state of a nucleic acid.

実施例に係る核酸の蛍光顕微鏡観察像である。It is a fluorescence microscope observation image of the nucleic acid which concerns on an Example.

実施例に係る核酸の蛍光顕微鏡観察像である。It is a fluorescence microscope observation image of the nucleic acid which concerns on an Example.

実施例で用いた蛍光顕微鏡の構成を簡略化して示す図である。It is a figure which simplifies and shows the structure of the fluorescence microscope used in the Example.

Claims (6)

蛍光特性部であるプテリジン構造体に核酸凝縮化機能部であるポリアミンを導入した下記の化１〜化７のいずれかに示す化学構造を有し、核酸の凝縮可視化剤として用いられることを特徴とする核酸処理剤。
It has a chemical structure shown in any one of the following chemical formulas 1 to 7 in which a polyamine that is a nucleic acid condensation functional part is introduced into a pteridine structure that is a fluorescent characteristic part, and is used as a nucleic acid condensation visualization agent Nucleic acid treating agent.
前記核酸処理剤が次の（１）及び／又は（２）の特性を有することを特徴とする請求項１に記載の核酸処理剤。
（１）ポリアミン部分による核酸凝縮化機能が、当該ポリアミンを単独の化学構造体として用いた場合に比較して、強化されている。
（２）核酸処理剤により凝縮可視化した核酸の凝縮状態が解除された際に、この核酸処理剤が核酸から脱離する。 The nucleic acid treating agent according to claim 1, wherein the nucleic acid treating agent has the following characteristics (1) and / or (2).
(1) The nucleic acid condensation function by the polyamine moiety is enhanced as compared with the case where the polyamine is used as a single chemical structure.
(2) When the condensed state of the nucleic acid visualized by condensation with the nucleic acid treatment agent is released, the nucleic acid treatment agent is detached from the nucleic acid. 前記核酸がＤＮＡであることを特徴とする請求項１又は請求項２に記載の核酸処理剤。 The nucleic acid treatment agent according to claim 1 or 2 , wherein the nucleic acid is DNA. 請求項１に規定する化１〜化７のいずれかに示す化学構造を有する核酸処理剤を核酸に作用させることにより、核酸を球（globule）状態に凝縮させる凝縮化と、球（globule）状態に凝縮化された核酸における蛍光特性部の高密度化に基づき、蛍光強度によって、凝縮化された核酸を十分に凝縮化されていない核酸から識別し又はバックグラウンドからの蛍光と識別するという核酸の可視化とを同時に発現させた核酸の凝縮可視化を行うことを特徴とする核酸の処理方法。 Condensation for condensing nucleic acid into a globule state by causing a nucleic acid treating agent having the chemical structure shown in any one of chemical formulas 1 to 7 defined in claim 1 to act on the nucleic acid, and a globule state the based-out densification of the fluorescence characteristic unit in the condenser of nucleic acids, the fluorescence intensity of identifying the fluorescence from the identification or background from a nucleic acid that is not sufficiently condensed the condensation of nucleic acids A method for treating nucleic acid, comprising condensing and visualizing nucleic acid expressed simultaneously with visualization of nucleic acid. 請求項１に規定する化１〜化７のいずれかに示す化学構造を有する核酸処理剤を核酸に作用させることにより、次の（３）、（４）を行うことを特徴とする核酸の処理方法。
（３）核酸を凝縮可視化する際には、核酸を球（globule）状態に凝縮させる凝縮化と、球（globule）状態に凝縮化された核酸における蛍光特性部の高密度化に基づき、蛍光強度によって、凝縮化された核酸を十分に凝縮化されていない核酸から識別し又はバックグラウンドからの蛍光と識別するという核酸の可視化とを同時に発現させた核酸の凝縮可視化を行い、
（４） in vitro 又は in vivoでの核酸の遺伝子操作又は遺伝子機能発現であるＰＣＲ法による遺伝子の増幅、細胞内での遺伝子融合又は遺伝子組替え、あるいはin vitro 又は in vivoでの遺伝子の転写又は複製のために核酸の凝縮状態を解除させる際には、前記遺伝子操作又は遺伝子機能発現に対する阻害因子であり得る蛍光特性部を核酸から脱離させる。 A nucleic acid treatment characterized by performing the following (3) and (4) by causing a nucleic acid treating agent having a chemical structure shown in any one of Chemical Formula 1 to Chemical Formula 7 defined in claim 1 to act on the nucleic acid: Method.
(3) when condensing visualize nucleic acids-out based nucleic acid and condensation of condensing the sphere (Globule) state, the density of the fluorescence characteristic portion of a sphere (Globule) state to the condensation of nucleic acids, Condensation visualization of the nucleic acid expressed simultaneously with the visualization of the nucleic acid that distinguishes the condensed nucleic acid from the nucleic acid that is not fully condensed by the fluorescence intensity or distinguishes it from the fluorescence from the background ,
(4) Gene amplification by PCR, which is genetic manipulation of nucleic acid or gene function expression in vitro or in vivo, gene fusion or gene recombination in cells, or transcription or replication of gene in vitro or in vivo In order to release the condensed state of the nucleic acid for the purpose, the fluorescent characteristic part which may be an inhibitor for the gene manipulation or gene function expression is detached from the nucleic acid. 前記核酸処理剤が細胞及び生物体に対して無毒性なものであることを特徴とする請求項４又は請求項５に記載の核酸の処理方法。 6. The method for treating a nucleic acid according to claim 4 or 5 , wherein the nucleic acid treatment agent is non-toxic to cells and organisms.
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