JP2005013023A - Method for fluorescent labeling of probe - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for labeling without using a DNA in which a double-stranded DNA comprising a bulge base present therein is labeled. <P>SOLUTION: The method for fluorescent labeling a probe is carried out as follows. A bulge base-recognizing molecule specifically forming a hydrogen bond with the bulge base and covalently binding to a DNA is reacted with the double-stranded DNA comprising the bulge base present therein to form a conjugate. The resultant conjugate is then chemically treated. A method for detecting, identifying or determining the bulge base by using the method is provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００１２】
（式中、Ａは、＝Ｃ−又は＝Ｎ−を示し、
Ｚは、−ＣＨ_２−又は−ＮＨ−を示し、
Ｒ_１は、水素原子、炭素数１〜１５のアルキル基であって当該アルキル基中の１個又はそれ以上の炭素原子が酸素原子、イオウ原子又は窒素原子で置換されてもよいアルキル基、炭素数１〜１５のアルコキシ基であって当該アルコキシ基中の１個又はそれ以上の炭素原子が酸素原子、イオウ原子又は窒素原子で置換されてもよいアルコキシ基、又は、炭素数１〜１５のモノ若しくはジアルキルアミノ基であって当該アルキルアミノ基中の１個又はそれ以上の炭素原子が酸素原子、イオウ原子又は窒素原子で置換されてもよいモノ若しくはジアルキルアミノ基を示し、Ｒ_２は、バルジ塩基と共有結合を形成し得る官能基を置換基として有する炭素数１〜２０のアルキル基であって当該アルキル基中の１個又はそれ以上の炭素原子が酸素原子、イオウ原子又は窒素原子で置換されてもよいアルキル基を示し、ｎは０もしくは１である。）
で表される化合物である。
【００１３】
本発明のバルジ塩基認識分子として、例えば、グアニンのバルジ塩基を認識するものとして、１，８−ナフチリジン誘導体から誘導された、次式（ＩＩ）、
【００１４】
【化３】

【００１５】
で表される２−（β−（Ｎ−エポキシメチル−アミノ）−プロピオニルアミノ）−７−メチル−１，８−ナフチリジンに基づいて図１を参照して本発明を説明する。
【００１６】
ターゲット遺伝子（一本鎖）にグアニン一塩基多い配列を持つプローブをハイブリダイズすると、グアニンバルジを持つ二本鎖が生成する（工程２）。このグアニンバルジ二本鎖ＤＮＡと式（ＩＩ）化合物を反応させると２−アシルナフチリジンがグアニンバルジを認識する（工程２）。次いで、式（ＩＩ）化合物の側鎖にあるエポキシドがターゲット及び／又はプローブのＤＮＡと共有結合（ラベル化）する（工程３）。この時点で二本鎖に２−アシルアミノナフチリジンが共有結合しているが、２−アシルアミノナフチリジンの蛍光は弱いので、検出することは難しい。次に、反応系より過剰の修飾分子（バルジ塩基認識分子）を除去し、２−アシルアミノナフチリジンで修飾された二本鎖ＤＮＡを酸で処理すると、２−アシルアミノナフチリジンのアミド結合が加水分解され、蛍光性の２−アミノナフチルジンが生成する（工程４）。
【００１７】
この蛍光を観測することにより、グアニンバルジの存在を判定することができる。ＤＮＡが修飾されていない場合、即ち、グアニンバルジが二本鎖ＤＮＡ上にない場合には、２−アミノナフチリジンが生成しないので、蛍光は観察されない。
【発明の実施の形態】
【００１８】
本発明のバルジ認識分子は、バルジ塩基認識部及びバルジ塩基修飾部を含有し、この両部が適当な炭素鎖、好ましくは当該炭素鎖中の１個又はそれ以上の炭素原子は酸素原子又は窒素原子で置換されていてもよい炭素鎖によって結合されていることを特徴とするものであり、かかる特徴を有するものであれば特に制限はない。本発明のバルジ認識分子の好ましいものとして一般式（Ｉ）で表される物質を挙げることができる。一般式（Ｉ）における置換基Ｒ_１は無くてもよいが（置換基Ｒ_１とが水素原子の場合）、置換基Ｒ_１としてはバルジ塩基を認識するために障害にならないものであれば特に制限はなく、例えば、炭素数１〜１５、好ましくは１〜１０より好ましくは１〜７の直鎖状又は分枝状のアルキル基、炭素数１〜１５、好ましくは１〜１０より好ましくは１〜７の直鎖状又は分枝状のアルキル基からなるアルコキシ基、炭素数１〜１５、好ましくは１〜１０より好ましくは１〜７の直鎖状又は分枝状のアルキル基でモノ又はジ置換されているモノ若しくはジアルキルアミノ基などが挙げられる。これらのアルキル基、アルコキシ基又はモノ若しくはジアルキルアミノ基における１個又はそれ以上の炭素原子は、酸素原子又は窒素原子で置換されていてもよい。置換基Ｒ_１は、ナフチリジン又はキノリン骨格のいずれの位置に存在してもよく、またこのような置換基が２個以上存在していてもよい。
【００１９】
本発明の一般式（Ｉ）で表されるバルジ認識分子における置換基Ｒ_２は、バルジ塩基と共有結合を形成し得る官能基を置換基として有する炭素数１〜２０、好ましくは炭素数３〜２０、炭素数３〜１０、又は炭素数３〜７のアルキル基であって当該アルキル基中の１個又はそれ以上の炭素原子が酸素原子又は窒素原子で置換されてもよいアルキル基を示し、好ましくは当該アルキル基中の少なくとも１個の炭素原子が窒素原子（−ＮＨ−）で置換された構造を有するものである。当該アルキル基は、さらに必要ならば、例えば分子の親水性を上げるためや、ＤＮＡなどとの親和性のためにある程度の極性を有する置換基を有してもよく、このような置換基は当該アルキル基の末端に位置していてもよいし、アルキル基の他の位置に存在していてもよいし、また２個以上の置換基が存在していてもよい。このような置換基としては、例えば、アミノ基、水酸基、カルボキシル基などが挙げられる。本発明のバルジ認識分子をチップなどの担体に固定化する場合には、当該アルキル基の任意の位置から担体に固定用のリンカー基を有してもよい。このようなリンカー基としては、アルキレン鎖や当該アルキレン鎖中の１個以上の炭素原子が窒素原子、酸素原子、硫黄原子、又はアミド結合などで置換されたものであってもよい。このようなリンカーの長さは特に制限はなく、担体に安定に固定するために必要な長さとすることができる。
【００２０】
また、一般式（Ｉ）における置換基Ｒ_２のバルジ塩基と共有結合を形成し得る官能基としては、室温または加温状態においてバルジ塩基と反応することができる反応性の基であれば特に制限はないが、エポキシ基、アジリジニル基、シクロプロピル基などの３員環の基が好ましい例として挙げられるが、他の反応性に基であってもよい。
さらに、一般式（Ｉ）におけるＡが窒素原子であるナフチリジン誘導体が好ましいが、Ａが炭素原子であるキノリン誘導体であってもよい。一般式（Ｉ）におけるＺがメチレン基の場合のアミド型であってもよいし、Ｚが窒素原子（−ＮＨ−）の場合の尿素型であってもよい。
本発明の一般式（Ｉ）で表されるバルジ認識分子の好ましい例としては、一般式（ＩＩ）で表されるエポキシナフチリジンが挙げられる。
【００２１】
本発明のバルジ認識分子、好ましくは一般式（Ｉ）で表されるバルジ塩基認識分子は低分子有機化合物であり、通常の有機合成法により適宜製造することができる。例えば、アミノナフチリジンやアミノキノリン誘導体のアミノ基をカルボン酸類や置換基を有するカルボン酸又はその反応性誘導体を用いてアミド化し、次いでカルボン酸部分にバルジ塩基と反応性の官能基を導入する方法により製造することができる。例えば、前記した一般式（ＩＩ）で表されるエポキシナフチリジンは、２−アミノ−１，８−ナフチリジン又は２−アミノ−７−メチル−１，８−ナフチリジンとＮ−保護−４−アミノ−酪酸又はＮ−保護−３−アミノ−プロピオン酸とを反応させてアミド誘導体とし、ついで末端のアミノ基の保護基を脱保護した後、エポキシ基を導入して製造することができる。
【００２２】
工程１における、グアニンバルジ形成は、一本鎖のターゲット遺伝子とグアニン一塩基多いプローブを通常の方法でハイブリダイズすればよい。例えば、ターゲット遺伝子とプローブを緩衝液中９５℃で短時間（５分）加熱後、ゆっくりと放冷することにより、グアニンバルジを形成することができる。
【００２３】
工程２及び３におけるグアニンバルジ二本鎖ＤＮＡとバルジ認識分子との反応は、緩衝溶液中で、二本鎖ＤＮＡとバルジ認識分子を反応させればよい。緩衝液としては、例えばカコジル緩衝液などが挙げられ、これらのｐＨは６．０〜８．０が好ましく、中性付近が特に好ましい。バルジ認識分子の使用量はは二本鎖ＤＮＡに対して１〜１００倍であり、好ましくは１０倍程度である。反応は、０〜６０℃、好ましくは４〜５０℃で、１０〜２４時間程度で終了する。
【００２４】
工程４における化学処理は、加溶媒分解が好ましく、特に酸による加水分解が特に好ましい。酸としては、塩酸、硫酸、硝酸などの無機酸、酢酸、ベンゼンスルホン酸などの有機酸が挙げられるが、濃塩酸の使用が特に好ましい。反応は例えば、工程３終了後に過剰のバルジ認識分子を例えば、エタノール沈殿、液体クロマトグラフィーや分子サイズ排除型のメンブランフィルターなどで除去し、次いで例えば濃塩酸を加えて、５０〜９５℃、好ましくは６０〜９０℃で、０．５〜３時間加熱することにより終了する。
【００２５】
上記の反応によって生成した２−アミノナフチリジンの濃度は例えば、蛍光スペクトルを励起波長３５２ｎｍで測定し、濃度既知の２−アミノナフチリジンを対照として用い、それらの強度比から求めることができる。
【００２６】
本発明の方法は、任意の配列のラベル化が可能であり、蛍光ラベルしたＤＮＡの合成が不要であるためコストが安くなり、さらに、ある条件に合致したＤＮＡ（グアニンバルジが形成されたＤＮＡ）のみがラベル化されるなどの特徴を有する、有用な方法である。
【００２７】
【実施例】
以下、実施例により本発明をより具体的に説明するが、本発明はこれら実施例により何ら限定されるものではない。
【００２８】
実施例１（エポキシナフチリジンの製造法）
エポキシナフチリジンの製造における反応式を次に示す。
【００２９】
【化４】

【００３０】
（１）３−アミノ−Ｎ−（７−メチルピリジノ［３，２−ｅ］ピリジン−２−イル）プロパンアミド（１．２３ｇ、５．３４ｍｍｏｌ）の無水ＣＨＣｌ_３（１５ｍＬ）溶液に（Ｓ）−（＋）−エピクロルヒドリン（０．６ｍＬ、７．６５ｍｍｏｌ）を加え、室温で２７．５時間撹件した。溶媒を濃縮留去した後、残留物をシリカゲルクロマトグラフィー（ＣＨＣｌ_３／ＭｅＯＨ＝１０：１）により精製し、目的の付加体（９５．４ｍｇ、０．２９ｍｍｏｌ、５．５％）を白色固体として得た。
^１Ｈ−ＮＭＲ （ＣＤ_３ＯＤ，４００ＭＨｚ） δ：
１１．１ （ｂｒ， １Ｈ）， ８．４３ （ｄ， １Ｈ， Ｊ＝８．８Ｈｚ）， ８．１１ （ｄ， １Ｈ， Ｊ＝８．８Ｈｚ），
８．００ （ｄ， １Ｈ， Ｊ＝８．０Ｈｚ）， ７．２７ （ｄ， １Ｈ， Ｊ＝８．０Ｈｚ）， ４．１３ （ｍ， １Ｈ），
３．６４ （ｍ， ２Ｈ）， ３．０８ （ｍ， ２Ｈ）， ２．９８ （ｄｄ， １Ｈ， Ｊ＝１２．８，３．２Ｈｚ）．
【００３１】
（２）続いて、水素化ナトリウム（６０％油分散液）（１９．８ｍｇ、０．４９５ｍｍｏｌ）の無水ＴＨＦ（０．６ｍＬ）溶液に、前記（１）で得た付加体（１２．５ｍｇ、３８．７μｍｏｌ）を加え、室温で１０分間撹件した。混合物を飽和ＮＨ_４Ｃｌ水溶液で希釈した後、ＣＨＣｌ_３で抽出した。有機層を無水ＭｇＳＯ_４で乾燥させ濃縮した。残留物をシリカゲルクロマトグラフィー（ＣＨＣｌ_３／ＭｅＯＨ＝１０：１）により精製し、目的のエポキシナフチリジン（８．６ｍｇ、３０．０μｍｏｌ、７８％）を白色固体として得た。
^１Ｈ−ＮＭＲ （ＣＤ_３ＯＤ，４００ＭＨｚ） δ：
８．４５ （ｄ， １Ｈ， Ｊ＝８．８Ｈｚ）， ８．１０ （ｄ， １Ｈ， Ｊ＝８．８Ｈｚ），
７．９８ （ｄ， １Ｈ， Ｊ＝８．０Ｈｚ）， ７．２５（ｄ， １Ｈ， Ｊ＝８．０Ｈｚ）， ３．２２ （ｍ， １Ｈ），
３．１５−３．０７ （ ３Ｈ）， ２．８１ （ｍ， １Ｈ）， ２．７４ （ｓ， ３Ｈ）， ２．６８−２．６５ （ ２Ｈ），
２．６０ （ｍ， ２Ｈ）
【００３２】
実施例２ （グアニンバルジＤＮＡとエボキシナフチリジンの反応）
実験に用いたオリゴマー（１）及びオリゴマー（２）の塩基配列を次に示す。
＊
オリゴマー（１） ５’−ＧＴＴＧＴＧＴＴＧＧＡ−３’
オリゴマー（２） ３’−ＣＡＡＣＡ ＡＡＣＣＴ−５’
このオリゴマー（２）は、オリゴマー（１）のグアニン（Ｇ）の位置（＊印を付した部分）の塩基が欠損しており、オリゴマー（１）のグアニン（Ｇ）がバルジ塩基となっている。
このオリゴマー（１）とオリゴマー（２）とをハイブリダイズさせたｄ（５’−ＧＴＴＧＴＧＴＴＧＧＡ−３’）／ｄ（３’−ＣＡＡＣＡ ＡＡＣＣＴ−５’）（６２．５μＭ，標準濃度）および内部標準としてｄＴ（１００μＭ）を含むカコジル酸緩衝溶液（５０ｍＭ，ｐＨ７．０）に、実施例１で製造したエポキシナフチリジン（１２５μＭ）を加え、４℃で１８時間反応させた。反応混合物をＨＰＬＣにより分析した。
分析条件：カラム ＣｈｃｍｃｏＢｏｎｄ ５−ＯＤＳ−Ｈ（４．６ｘ１５０ｍｍ）、溶出 ０．１Ｍ ＴＥＡＡバッファー、７−３０％アセトニトリル直線勾配、０−３０分、溶出速度 １．０ｍＬ／分。
生成物のＨＰＬＣピークは２５４ｎｍで観測した。２０時間後付加体を分取し、ＭＡＬＤＩ−ＴＯＦ ＭＳ測定により分析した。
オリゴマー（２）（Ａ Ａ）とオリゴマー（１）（ＴＧＴ）からなるグアニンバルジをエポキシナフチリジン（図中の２）と反応させ４℃１８時間後のＨＰＬＣ分析結果を図２に示す。オリゴマー（１）と２の付加体（２−ＴＧＴ）の生成と共にＴＧＴの減少が認められた。カッコ内の数字は反応スタート時における各オリゴマーの内部標準ｄＴ（デオキシチミジン）に対するＨＰＬＣのピーク面積との差を％で表示している。オリゴマー（２）はほとんど減少していない。
【００３３】
実施例３ （加水分解）
実施例２で得られた反応混合物をＨＰＬＣで過剰のバルジ認識分子を除き精製、濃縮し、これに濃塩酸１ｍｌを加え、１５℃で４０分間加熱し、濃縮した。
【００３４】
実施例４ （蛍光スペクトルの測定）
（ａ）実施例３で得られた粗組成物を７５０ｍｌのバッファーに溶解した溶液、（ｂ）同緩衝液に溶解した２−アミノナフチリジン（１０μＭ）及び（ｃ）化合物（ＩＩ）（１０μＭ）をカコジル酸緩衝溶液（１０ｍＭ、ｐＨ７．０）中で励起波長３５３ｎｍで測定した。結果を図３に示す。
（ａ）と（ｂ）の蛍光強度比から２−アミノナフチリジンの濃度が３７μＭ（２７ｎｍｏｌ）と計算された。
【００３５】
【発明の効果】
本発明の蛍光標識化方法は、蛍光標識化したＤＮＡを用いずにバルジが形成されたＤＮＡのみを標識化するもので、操作が容易で、安価な方法である。この方法を用いることにより、遺伝子の欠損の有無や、欠損した塩基の種類を高感度で、大量かつ迅速に検出、同定又は定量することができることになり、ＤＮＡの損傷に伴う各種疾患の治療、予防又は診断に有用である。
【図面の簡単な説明】
【図１】図１は、本発明方法の概要を模式的に示したものである。
【図２】図２は、実施例２で得られた複合体のＨＰＬＣのチャートを示す。
【図３】図３は、実施例４で得られた蛍光スペクトルを示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for fluorescently labeling a DNA probe in which a bulge base is present, and a method for measuring the presence or absence of a bulge base using the same.
[0002]
[Prior art]
Bulge structures present in DNA and RNA play an important role in the recognition of nucleic acids by proteins. Molecules that specifically bind to these structures are attracting attention from the viewpoint of drug development because they have the potential as inhibitors of proteins that recognize the bulge structure.
A bulge DNA recognition molecule is a molecule that specifically binds to and stabilizes DNA (bulge DNA) having an unpaired base (bulge base) generated in double-stranded DNA. The bulge DNA targeted by this recognition molecule occurs as a result of DNA replication errors or DNA damage. In addition, the presence or absence of a bulge base, which is a genetic abnormality, is used for diagnosis of genetic diseases and the like. Therefore, this bulge DNA recognition molecule can be used as 1) a diagnostic agent for examining the presence or absence of a gene defect, 2) a DNA damage detection agent, 3) a gene damage stabilizer, 4) a DNA repair enzyme inhibitor. Not only is it expected, it is an important substance in research and development such as gene damage and gene duplication mistakes.
[0003]
The present inventors have previously described a bulge base recognition molecule that can specifically form a hydrogen bond with a bulge base and can be stacked by a base pair existing in the vicinity of the bulge base and stably incorporated into a duplex. The bulge recognition molecule was further found to have a functional group capable of forming a covalent bond with the bulge base (Patent Document 1).
[0004]
This bulge recognition molecule recognizes the basil base of double-stranded DNA, and then covalently binds to this to form a stable complex. Therefore, the bulge recognition molecule contains the bulge base by immobilizing the bulge recognition molecule. Only DNA can be selectively immobilized, and it has become possible to detect, identify or quantify DNA as immobilized using a carrier such as a chip.
[0005]
However, in order to actually measure the bulge base, complicated steps such as immobilizing the bulge recognition molecule on a carrier such as a chip and labeling any of the DNAs to be assayed are necessary and costly. It was a thing.
[0006]
[Patent Document 1] Japanese Patent Application No. 2002-62883
[Problems to be solved by the invention]
The present invention relates to a method of fluorescently labeling a probe by reacting a double-stranded DNA in which a bulge base is present and the above bulge base recognition molecule to form a complex, and chemically treating the complex. The present invention provides a method for easily detecting and identifying a bulge base.
[0008]
[Means for Solving the Problems]
In order to solve the above problems, the present inventors treated a complex obtained by reacting a double-stranded DNA in which a bulge base is present with the above bulge base recognition molecule with an acid. The product cannot be obtained by reacting double-stranded DNA with no bulge base and the above bulge base recognition molecule, so that even if it is treated with acid, no fluorescent substance is produced. I found.
[0009]
That is, the present invention is a method of hybridizing a DNA having a normal base sequence and a sample DNA, reacting this with a bulge base recognition molecule, treating the resulting complex with an acid, and fluorescently labeling this, The present invention relates to a method for determining whether or not a subject forms a bulge base by the method.
[0010]
As described in Japanese Patent Application No. 2000-62883, the bulge base recognition molecule used in the present invention has the following general formula (I),
[0011]
[Chemical 2]

[0012]
(In the formula, A represents = C- or = N-,
Z represents —CH ₂ — or —NH—;
R ₁ is a hydrogen atom, an alkyl group having 1 to 15 carbon atoms, and one or more carbon atoms in the alkyl group may be substituted with an oxygen atom, a sulfur atom or a nitrogen atom, carbon An alkoxy group having 1 to 15 carbon atoms, wherein one or more carbon atoms in the alkoxy group may be substituted with an oxygen atom, a sulfur atom or a nitrogen atom, or a monovalent carbon atom having 1 to 15 carbon atoms Or a dialkylamino group, which represents a mono- or dialkylamino group in which one or more carbon atoms in the alkylamino group may be substituted with an oxygen atom, a sulfur atom or a nitrogen atom, and R ₂ represents a bulge base An alkyl group having 1 to 20 carbon atoms having a functional group capable of forming a covalent bond with the substituent, wherein one or more carbon atoms in the alkyl group are oxygen atoms, Substituted by Au atom or a nitrogen atom represents an optionally alkyl group, n represents 0 or 1. )
It is a compound represented by these.
[0013]
As the bulge base recognition molecule of the present invention, for example, as a molecule that recognizes the bulge base of guanine, the following formula (II) derived from a 1,8-naphthyridine derivative,
[0014]
[Chemical 3]

[0015]
The present invention will be described with reference to FIG. 1 based on 2- (β- (N-epoxymethyl-amino) -propionylamino) -7-methyl-1,8-naphthyridine represented by:
[0016]
When a probe having a guanine single nucleotide-rich sequence is hybridized to the target gene (single strand), a double strand having a guanine bulge is generated (step 2). When this guanine bulge double-stranded DNA is reacted with the compound of the formula (II), 2-acylnaphthyridine recognizes guanine bulge (step 2). Next, the epoxide on the side chain of the compound of formula (II) is covalently bonded (labeled) to the target and / or probe DNA (step 3). At this point, 2-acylaminonaphthyridine is covalently bound to the double strand, but the fluorescence of 2-acylaminonaphthyridine is weak and difficult to detect. Next, excess modified molecules (bulge base recognition molecules) are removed from the reaction system, and the double-stranded DNA modified with 2-acylaminonaphthyridine is treated with an acid, whereby the amide bond of 2-acylaminonaphthyridine is hydrolyzed. As a result, fluorescent 2-aminonaphthyridine is produced (step 4).
[0017]
By observing this fluorescence, the presence of guanine bulge can be determined. If the DNA is not modified, i.e., if the guanine bulge is not on the double-stranded DNA, no 2-aminonaphthyridine is formed and no fluorescence is observed.
DETAILED DESCRIPTION OF THE INVENTION
[0018]
The bulge recognition molecule of the present invention contains a bulge base recognition portion and a bulge base modification portion, both of which are suitable carbon chains, preferably one or more carbon atoms in the carbon chain are oxygen atoms or nitrogen. It is characterized by being bonded by a carbon chain which may be substituted with an atom, and there is no particular limitation as long as it has such a feature. Preferred examples of the bulge recognition molecule of the present invention include substances represented by general formula (I). The substituent R ₁ in the general formula (I) may be omitted (when the substituent R ₁ is a hydrogen atom), and the substituent R ₁ is not particularly limited as long as it does not interfere with the recognition of the bulge base. There is no restriction, for example, a linear or branched alkyl group having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 7 carbon atoms, 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably 1 An alkoxy group consisting of a linear or branched alkyl group of ˜7, mono- or di-C 1-15, preferably 1-10, more preferably 1-7 linear or branched alkyl group. Examples thereof include a substituted mono- or dialkylamino group. One or more carbon atoms in these alkyl group, alkoxy group or mono- or dialkylamino group may be substituted with an oxygen atom or a nitrogen atom. The substituent R ₁ may be present at any position of the naphthyridine or quinoline skeleton, and two or more such substituents may be present.
[0019]
The substituent R ₂ in the bulge recognition molecule represented by the general formula (I) of the present invention has 1 to 20 carbon atoms, preferably 3 to 3 carbon atoms, having a functional group capable of forming a covalent bond with a bulge base as a substituent. 20, an alkyl group having 3 to 10 carbon atoms, or an alkyl group having 3 to 7 carbon atoms, in which one or more carbon atoms in the alkyl group may be substituted with an oxygen atom or a nitrogen atom, Preferably, it has a structure in which at least one carbon atom in the alkyl group is substituted with a nitrogen atom (—NH—). If necessary, the alkyl group may have a substituent having a certain degree of polarity, for example, for increasing the hydrophilicity of the molecule or for affinity with DNA. It may be located at the terminal of the alkyl group, may be present at other positions of the alkyl group, or two or more substituents may be present. Examples of such a substituent include an amino group, a hydroxyl group, and a carboxyl group. When the bulge recognition molecule of the present invention is immobilized on a carrier such as a chip, it may have a linker group for immobilization on the carrier from any position of the alkyl group. As such a linker group, an alkylene chain or one or more carbon atoms in the alkylene chain may be substituted with a nitrogen atom, an oxygen atom, a sulfur atom, an amide bond or the like. The length of such a linker is not particularly limited, and can be a length necessary for stably fixing to a carrier.
[0020]
Further, the functional group capable of forming a covalent bond with the bulge base of the substituent R ₂ in the general formula (I) is not particularly limited as long as it is a reactive group capable of reacting with the bulge base at room temperature or in a heated state. Although preferred examples include 3-membered ring groups such as an epoxy group, aziridinyl group, and cyclopropyl group, other reactive groups may also be used.
Further, a naphthyridine derivative in which A in the general formula (I) is a nitrogen atom is preferable, but a quinoline derivative in which A is a carbon atom may be used. It may be an amide type when Z in the general formula (I) is a methylene group, or a urea type when Z is a nitrogen atom (—NH—).
Preferable examples of the bulge recognition molecule represented by the general formula (I) of the present invention include epoxy naphthyridine represented by the general formula (II).
[0021]
The bulge recognition molecule of the present invention, preferably the bulge base recognition molecule represented by the general formula (I), is a low-molecular organic compound, and can be appropriately produced by ordinary organic synthesis methods. For example, the amino group of aminonaphthyridine or aminoquinoline derivative is amidated using a carboxylic acid, a carboxylic acid having a substituent or a reactive derivative thereof, and then a functional group reactive with a bulge base is introduced into the carboxylic acid moiety. Can be manufactured. For example, the epoxy naphthyridine represented by the general formula (II) is 2-amino-1,8-naphthyridine or 2-amino-7-methyl-1,8-naphthyridine and N-protected-4-amino-butyric acid. Alternatively, it can be produced by reacting with N-protected-3-amino-propionic acid to obtain an amide derivative and then deprotecting the protecting group of the terminal amino group, and then introducing an epoxy group.
[0022]
The guanine bulge formation in step 1 may be performed by hybridizing a single-stranded target gene and a probe having one guanine single base by a usual method. For example, a guanine bulge can be formed by heating the target gene and probe in a buffer solution at 95 ° C. for a short time (5 minutes) and then slowly cooling them.
[0023]
The reaction between the guanine bulge double-stranded DNA and the bulge recognition molecule in the steps 2 and 3 may be performed by reacting the double-stranded DNA with the bulge recognition molecule in a buffer solution. Examples of the buffer solution include a cacodyl buffer solution, and the pH thereof is preferably 6.0 to 8.0, and particularly preferably near neutral. The amount of bulge recognition molecule used is 1 to 100 times, preferably about 10 times that of double-stranded DNA. The reaction is carried out at 0 to 60 ° C., preferably 4 to 50 ° C., for about 10 to 24 hours.
[0024]
The chemical treatment in step 4 is preferably solvolysis, particularly preferably hydrolysis with an acid. Examples of the acid include inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid, and organic acids such as acetic acid and benzenesulfonic acid, but the use of concentrated hydrochloric acid is particularly preferable. In the reaction, for example, after completion of step 3, excess bulge recognition molecules are removed by, for example, ethanol precipitation, liquid chromatography, molecular size exclusion type membrane filter, etc., and then concentrated hydrochloric acid is added, for example, at 50 to 95 ° C., preferably It is completed by heating at 60 to 90 ° C. for 0.5 to 3 hours.
[0025]
The concentration of 2-aminonaphthyridine produced by the above reaction can be determined, for example, by measuring the fluorescence spectrum at an excitation wavelength of 352 nm, using 2-aminonaphthyridine having a known concentration as a control, and determining the intensity ratio thereof.
[0026]
The method of the present invention can label any sequence, eliminates the need for the synthesis of fluorescently labeled DNA, reduces the cost, and further meets a certain condition (DNA with a guanine bulge formed). This is a useful method with features such as only being labeled.
[0027]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.
[0028]
Example 1 (Production method of epoxy naphthyridine)
The reaction formula in the production of epoxy naphthyridine is shown below.
[0029]
[Formula 4]

[0030]
(1) To a solution of 3-amino-N- (7-methylpyridino [3,2-e] pyridin-2-yl) propanamide (1.23 g, 5.34 mmol) in anhydrous CHCl ₃ (15 mL), (S) — (+)-Epichlorohydrin (0.6 mL, 7.65 mmol) was added and stirred at room temperature for 27.5 hours. After distilling off the solvent, the residue was purified by silica gel chromatography (CHCl ₃ / MeOH = 10: 1) to give the desired adduct (95.4 mg, 0.29 mmol, 5.5%) as a white solid. Obtained.
¹ H-NMR (CD ₃ OD, 400 MHz) δ:
11.1 (br, 1H), 8.43 (d, 1H, J = 8.8 Hz), 8.11 (d, 1H, J = 8.8 Hz),
8.00 (d, 1H, J = 8.0 Hz), 7.27 (d, 1H, J = 8.0 Hz), 4.13 (m, 1H),
3.64 (m, 2H), 3.08 (m, 2H), 2.98 (dd, 1H, J = 12.8, 3.2 Hz).
[0031]
(2) Subsequently, sodium hydride (60% oil dispersion) (19.8 mg, 0.495 mmol) in anhydrous THF (0.6 mL) was added to the adduct (12.5 mg, 38.7 μmol) was added and stirred at room temperature for 10 minutes. The mixture was diluted with saturated aqueous NH ₄ Cl and then extracted with CHCl ₃ . The organic layer was dried over anhydrous MgSO ₄ and concentrated. The residue was purified by silica gel chromatography (CHCl ₃ / MeOH = 10: 1) to give the desired epoxy naphthyridine (8.6 mg, 30.0 μmol, 78%) as a white solid.
¹ H-NMR (CD ₃ OD, 400 MHz) δ:
8.45 (d, 1H, J = 8.8 Hz), 8.10 (d, 1H, J = 8.8 Hz),
7.98 (d, 1H, J = 8.0 Hz), 7.25 (d, 1H, J = 8.0 Hz), 3.22 (m, 1H),
3.15-3.07 (3H), 2.81 (m, 1H), 2.74 (s, 3H), 2.68-2.65 (2H),
2.60 (m, 2H)
[0032]
Example 2 (Reaction of guanine bulge DNA and eboxynaphthyridine)
The base sequences of oligomer (1) and oligomer (2) used in the experiment are shown below.
*
Oligomer (1) 5′-GTTGGTTGGA-3 ′
Oligomer (2) 3′-CAACA AACCT-5 ′
In this oligomer (2), the base at the position of guanine (G) in the oligomer (1) (the part marked with *) is missing, and the guanine (G) in the oligomer (1) is a bulge base. .
D (5′-GTTGGTTGGA-3 ′) / d (3′-CAACA AACCT-5 ′) (62.5 μM, standard concentration) hybridized with the oligomer (1) and the oligomer (2) and an internal standard Epoxynaphthyridine (125 μM) produced in Example 1 was added to a cacodylate buffer solution (50 mM, pH 7.0) containing dT (100 μM) and reacted at 4 ° C. for 18 hours. The reaction mixture was analyzed by HPLC.
Analysis conditions: Column ChcmcoBond 5-ODS-H (4.6 × 150 mm), elution 0.1 M TEAA buffer, 7-30% acetonitrile linear gradient, 0-30 minutes, elution rate 1.0 mL / min.
The product HPLC peak was observed at 254 nm. After 20 hours, the adduct was separated and analyzed by MALDI-TOF MS measurement.
Oligomer (2) (A A guanine bulge composed of A) and oligomer (1) (TGT) is reacted with epoxynaphthyridine ( 2 in the figure), and the HPLC analysis result after 18 hours at 4 ° C. is shown in FIG. Reduction of TGT along with generation of the oligomer (1) and 2 adduct (2-TGT) was found. The numbers in parentheses indicate the difference in percentage from the peak area of HPLC with respect to the internal standard dT (deoxythymidine) of each oligomer at the start of the reaction. The oligomer (2) is hardly decreased.
[0033]
Example 3 (Hydrolysis)
The reaction mixture obtained in Example 2 was purified by HPLC after removing excess bulge recognition molecules, and 1 ml of concentrated hydrochloric acid was added thereto, heated at 15 ° C. for 40 minutes, and concentrated.
[0034]
Example 4 (Measurement of fluorescence spectrum)
(A) a solution obtained by dissolving the crude composition obtained in Example 3 in 750 ml of buffer, (b) 2-aminonaphthyridine (10 μM) and (c) compound (II) (10 μM) dissolved in the same buffer. Measurement was carried out in a cacodylate buffer solution (10 mM, pH 7.0) at an excitation wavelength of 353 nm. The results are shown in FIG.
From the fluorescence intensity ratio of (a) and (b), the concentration of 2-aminonaphthyridine was calculated to be 37 μM (27 nmol).
[0035]
【The invention's effect】
The fluorescent labeling method of the present invention is a method that labels only DNA in which bulges are formed without using fluorescently labeled DNA, and is easy to operate and inexpensive. By using this method, the presence or absence of a gene defect and the type of the deleted base can be detected, identified or quantified rapidly and in large quantities, and various diseases associated with DNA damage can be treated. Useful for prevention or diagnosis.
[Brief description of the drawings]
FIG. 1 schematically shows the outline of the method of the present invention.
FIG. 2 shows a HPLC chart of the complex obtained in Example 2.
FIG. 3 shows the fluorescence spectrum obtained in Example 4.

Claims (12)

A fluorescent labeling method for a probe, characterized in that a double-stranded DNA having a bulge base is reacted with a bulge base recognition molecule to form a complex, which is chemically treated. The fluorescent labeling method according to claim 1, wherein the chemical treatment is solvolysis. The fluorescent labeling method according to claim 1 or 2, wherein the double-stranded DNA has a guanine bulge obtained by hybridizing the DNA of the subject and a probe DNA having one more guanine base than the DNA. In the bulge base recognition molecule, the bulge recognition molecule can form a hydrogen bond specifically with the bulge base, and can be stacked by a base pair existing in the vicinity of the bulge base and stably incorporated into the duplex. 2. The fluorescence labeling method according to claim 1, wherein the bulge recognition molecule is a compound further having a functional group capable of forming a covalent bond with DNA. A bulge base recognition molecule is represented by the following general formula (I):

(In the formula, A represents = C- or = N-,
Z represents —CH ₂ — or —NH—;
R ₁ is a hydrogen atom, an alkyl group having 1 to 15 carbon atoms, and one or more carbon atoms in the alkyl group may be substituted with an oxygen atom, a sulfur atom or a nitrogen atom, carbon An alkoxy group having 1 to 15 carbon atoms, wherein one or more carbon atoms in the alkoxy group may be substituted with an oxygen atom, a sulfur atom or a nitrogen atom, or a monovalent carbon atom having 1 to 15 carbon atoms Or a dialkylamino group, which represents a mono- or dialkylamino group in which one or more carbon atoms in the alkylamino group may be substituted with an oxygen atom, a sulfur atom or a nitrogen atom, and R ₂ represents a bulge base An alkyl group having 1 to 20 carbon atoms having a functional group capable of forming a covalent bond with the substituent, wherein one or more carbon atoms in the alkyl group are oxygen atoms, Substituted by Au atom or nitrogen atom shown an alkyl group which may, n represents 0 or 1. )
The fluorescent labeling method according to claim 1 or 4, wherein the compound is represented by the formula: 6. The fluorescent labeling method according to claim 1, wherein the bulge base recognition molecule is a 1,8-naphthyridine derivative. 5. The fluorescent labeling method according to claim 1, wherein the functional group capable of forming a covalent bond with DNA in the bulge base recognition molecule is an epoxy group. . The fluorescence labeling method according to claim 1 or 4, wherein the bulge base recognition molecule is 2- (β- (N-epoxymethyl-amino) -propionylamino) -7-methyl-1,8-naphthyridine. . The fluorescent labeling method according to claim 1, wherein the bulge base is guanine. The fluorescent labeling method according to claim 1, wherein the bulge base recognition molecule is a guanine bulge modified molecule. A probe DNA capable of forming a bulge base is hybridized with the sample DNA to form a double-stranded DNA, and a bulge base recognition molecule is reacted with the double-stranded DNA to form a complex. A method for detecting, identifying or quantifying a bulge base, which comprises subjecting a probe to fluorescent labeling after chemical treatment and measuring fluorescence. The method according to claim 11, wherein the chemical treatment is solvolysis.

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