JP4177006B2 - Guanimbulge modifying molecule - Google Patents

Description

【００１０】
（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又は窒素原子で置換されてもよいアルキル基を示す。）
で表される化合物であるバルジ塩基認識分子に関する。本発明のバルジ認識分子は、さらにこれをチップなどの担体に結合させるためのリンカー基を有することができる。また、本発明は、このようなリンカー基により担体に固定化されたバルジ認識分子に関する。
【００１１】
また、本発明は、前記した本発明のバルジ塩基認識分子を含有してなるバルジ塩基認識用組成物、並びに前記した本発明のバルジ塩基認識分子を用いて、ＤＮＡ中のバルジ塩基を検出、同定又は定量する方法及びそのための測定用キットに関する。
さらに、本発明は、バルジ塩基認識分子が、特定のバルジ塩基と水素結合を形成し、当該バルジ塩基の近隣に存在する塩基対にスタックされることによりバルジ塩基が安定化されたＤＮＡに関する。
【００１２】
本発明者らは、グアニン（Ｇ）のバルジ認識分子として１，８−ナフチリジン誘導体を開発してきた（特開２００１−８９４７８号）が、この分子に基づいてバルジ塩基との共有結合を形成するための手法を種々検討してきた。その結果、バルジ認識分子のバルジ塩基の認識部位と適当な距離を隔てて反応性の官能基を設けることによりバリジ塩基と共有結合を形成することができることを見出した。本発明のバルジ塩基と共有結合できるバルジ認識分子として、例えば、１，８−ナフチリジン誘導体から誘導された、次式（II）、
【００１３】
【化３】

【００１４】
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本発明を説明する。
本発明のバルジ認識分子は、図４に示されるようにバルジ塩基認識部（この例ではグアニン認識部）とバルジ塩基修飾部（この例ではグアニン修飾部）とからなり、図５のモデル図に示されるように、まずバルジ塩基認識部によりバルジ塩基を認識し、バルジ塩基認識部とバルジ塩基とが水素結合を形成する。次いで側鎖の活性水素がバルジ塩基の極性部分に基づいてバルジ塩基の方向に折れ曲がり、その結果、バルジ認識分子の反応性の官能基とバルジ塩基とが化学反応を起こすものと考えられる。
【００１５】
このことを次のオリゴマー（１）及びオリゴマー（２）を用いて確認した。
＊
オリゴマー（１） ５’−ＡＴＴＧＣＧＣＴＧＡ−３’
オリゴマー（２） ３’−ＴＡＡＣＧ ＧＡＣＴ−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）、オリゴマー（２）及び前記式（II）の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（以下、本明細書においては「エポキシナフチリジン」という。）をカコジル酸緩衝溶液中で室温で反応させた。反応前の高速液体クロマトグラフィー（ＨＰＬＣ）及び室温で２０時間後のＨＰＬＣ（内部標準としてｄＴを用いている。）の結果のチャートを図６に示す。
反応前（図６の上段のチャート）では、チャートの左側から内部標準のｄＴ、オリゴマー（２）、オリゴマー（１）及びエポキシナフチリジンの各ピークが観察されたが、反応後（図６の下段のチャート）では、チャートの左側から内部標準のｄＴ、オリゴマー（２）の大きなピーク、オリゴマー（１）の小さなピーク、エポキシナフチリジンの各ピークのさらに右側の新たな生成物の大きなピークが観察された。この新たな生成物のピークは、オリゴマー（１）のピークが小さくなっていることから、オリゴマー（１）とエポキシナフチリジンとの反応生成物であることが確認された。
この結果、オリゴマー（１）とエポキシナフチリジンとの反応率は約９７％であり、オリゴマー（２）との反応率は約３％であることがわかった。
【００１６】
本発明のバルジ認識分子によれば、バルジ塩基と共有結合を形成して、バルジ認識分子とバルジ塩基を含むＤＮＡが強固に結合していることから、バルジ認識分子を固定化することによりバルジ塩基を含んでいるＤＮＡのみを選択的に固定化することができ、チップなどの担体を用いて固定化したＤＮＡとして検出、同定又は定量化することが可能となる。例えば、ある遺伝子が１個又は数個の塩基を欠損しているか否かを検定する場合には、欠損箇所を含む当該遺伝子の断片を被検体遺伝子断片として用意し、当該断片を正常な塩基配列を有する正常遺伝子断片とハイブリダイズさせて、本発明のバルジ認識分子の存在下に反応を行うことにより、被検体遺伝子断片に塩基の欠損が有るか無いかを検出、同定又は定量することできる。
【００１７】
したがって、本発明のバルジ塩基認識分子は、これをバルジ塩基認識剤として使用することができ、また、適当な担体又は測定用の資材と組み合わせることによりバルジ塩基認識用組成物とすることができる。
また、本発明は、本発明のバルジ塩基認識分子又は標識化若しくは固定化されたバルジ塩基認識分子を使用してＤＮＡ中のバルジ塩基を検出、同定又は定量するための測定方法を提供するものである。本発明の測定方法としては、前記してきたＨＰＬＣの測定でもよいが、本発明のバルジ認識分子をチップ上などに固定化して検定するＤＮＡのいずれかを標識化して、チップ上などの担体上に残った標識物を測定することもできるが、これらの測定方法に限定されるものではない。また、測定に使用する遺伝子の断片としては特に制限はないが、好ましくは１０〜１００塩基、より好ましくは１０〜５０塩基、１０〜３０塩基程度の長さのものがよい。
さらに、このようなバルジ塩基の検定の際に、本発明者らが開発きたミスマッチ認識分子（特開２００１−１４９０９６号参照）を併せて使用することもできる。このようなミスマッチ認識分子を併せて使用することにより、被検体遺伝子断片における欠損塩基の有無及び種類を検定できると同時に、ＳＮＰのような塩基の変化についても検定することができる。
【００１８】
また、本発明のバルジ塩基認識分子はこれを単独で使用することもできるが、分子中の適当な位置に放射性元素を導入したり、化学発光又は蛍光を発する分子種を導入するなどして、標識化して使用することもできる。さらに、本発明のバルジ塩基認識分子の適当な位置においてポリスチレンなどの高分子材料やチップ上などに直接又はリンカーなどを用いて結合させて、これを固定化して使用することもできる。固定化して使用する場合には、グアニン（Ｇ）のバルジ塩基を選択的に判定することができるバルジ認識分子、シトシン（Ｃ）のバルジ塩基を選択的に判定することができるバルジ認識分子、チミン（Ｔ）のバルジ塩基を選択的に判定することができるバルジ認識分子、及びアデニン（Ａ）のバルジ塩基を選択的に判定することができるバルジ認識分子を並べて置くことにより、４種類の塩基のバルジの存在を一度の検定により判定することも可能となる。
【００１９】
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。
【００２０】
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。
【００２１】
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。
【００２２】
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。
【００２３】
本発明のバルジ塩基認識分子を用いることにより、１個又は２個以上のバルジ塩基を有するＤＮＡにおいて、特定の塩基によるバルジ塩基と水素結合を形成させてこれを安定化させ、さらにバルジ塩基と共有結合を形成することによりバルジ塩基を含有するＤＮＡを安定化させることができる。特に本発明のバルジ塩基認識分子は、特定のバルジ塩基と水素結合を形成し、かつ共有結合を形成するのみならず、近傍、好ましくは隣接する塩基対にスタックされ、バルジ塩基が存在しているにもかかわらず比較的安定なＤＮＡを得ることができる。
したがって、本発明は、バルジ塩基認識分子が、特定のバルジ塩基と水素結合を形成し、かつ共有結合を形成し、当該バルジ塩基の近隣に存在する塩基対にスタックされることによりバルジ塩基が安定化されたバルジ塩基を含有するＤＮＡを提供するものである。
本発明のＤＮＡは、バルジ塩基が本発明のバルジ塩基認識分子と水素結合により塩基対と同様な「対」を形成し、かつバルジ塩基と「対」を形成している本発明のバルジ塩基認識分子が近傍、好ましくは隣接の塩基対を形成している塩基にサンドイッチ状に挟まれてスタックされているものと考えられる。
【００２４】
本発明のバルジＤＮＡ認識分子を用いることにより、従来の技術では達成できないバルジＤＮＡ認識分子を高感度で、かつ固定化が可能となるために大量に迅速に検出、同定又は定量することができ、バルジ塩基の存在の有無や特定のバルジ塩基のみを選択して検出、同定又は定量できることから、本発明のバルジ認識分子はＤＮＡ損傷に伴う各種疾患の治療、予防又は診断に有用である。
また、本発明のＤＮＡはバルジ塩基を有した状態で比較的安定に存在することができることから、バルジ塩基を含有するＤＮＡの安定化や、バルジ塩基の発生原因やバルジ塩基の修復機構の解明などの研究材料としても重要である。
【００２５】
【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。
【００２６】
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。
【００２７】
【化４】

【００２８】
（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)．
【００２９】
（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)
【００３０】
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’−ＡＴＴＧＣＧＣＴＧＡ−３’
オリゴマー（２） ３’−ＴＡＡＣＧ ＧＡＣＴ−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（ＴＣＡＧ ＧＣＡＡＴ）／（ＡＴＴＧＣＧＣＴＧＡ）（５０μＭ，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で製造したエボキシナフチリジン（５００μＭ）を加え、室温で反応させた。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。
ＨＰＬＣの結果を図６に示す。
【００３１】
【発明の効果】
本発明のバルジ認識分子は、特定のバルジ塩基と水素結合を形成し、かつ共有結合を形成するのみならず、近傍の塩基対に安定にスタックされ、特定のバルジ塩基と共有結合により強力に結合できるものであることから、バルジ塩基を含むＤＮＡを本発明のバルジ認識分子を用いて固定化することも可能となる。そして、チップなどの担体に本発明のバルジ認識分子を固定化することにより、特定のバルジ塩基を含むＤＮＡのみを選択的に担体上に固定化することが可能となり、遺伝子の欠損の有無や欠損した塩基の種類を高感度で、大量にかつ迅速に検出、同定又は定量することができることになり、ＤＮＡ損傷に伴う各種疾患の治療、予防又は診断に極めて有用である。
【図面の簡単な説明】
【図１】図１は、塩基対の内側に向いているバルジ塩基（図ではグアニン）を模式的に示したものである。
【図２】図２は、塩基対の外側に向いているバルジ塩基（図ではグアニン）を模式的に示したものである。
【図３】図３は、塩基対の外側に向いているバルジ塩基（図ではグアニン）に、塩基対の外側からインターカーレーションする様子を模式的に示したものである。
【図４】図４は、本発明のバルジ認識分子の構成部分を例示した化合物に基づいて説明するものである。
【図５】図５は、本発明のバルジ認識分子がバルジ塩基を認識し、バルジ塩基と水素結合を形成し、次いでバルジ認識分子のバルジ塩基修飾部とバルジ塩基とが反応して共有結合を形成する様子をモデル化して例示したものである。
【図６】図６は、本発明のエポキシナフチリジンとバルジ塩基を含むＤＮＡとの反応の前（図６上段のチャート）と反応後（図６下段のチャート）ＨＰＬＣのチャートを示す。反応前（図６の上段のチャート）では、チャートの左側から内部標準のｄＴ、オリゴマー（２）、オリゴマー（１）及びエポキシナフチリジンの各ピークを示し、反応後（図６の下段のチャート）では、チャートの左側から内部標準のｄＴ、オリゴマー（２）、オリゴマー（１）、エポキシナフチリジン、及び新たな生成物の各ピークを示す。[0001]
BACKGROUND OF THE INVENTION
The present invention measures a bulge base recognition molecule capable of specifically recognizing a bulge base in DNA and capable of covalently binding to a bulge base, a composition for bulge base recognition, and a bulge base using the same. On how to do. The present invention also relates to DNA in which a bulge base is stabilized by forming a hydrogen bond with the bulge base and forming a covalent bond with the bulge base.
[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]
FIG. 1 shows an example of a bulge base. In this example, guanine (G) is in a state where it cannot form a base pair as a bulge base. The guanine (G) that is the bulge base in FIG. 1 is not hydrogen-bonded to any base, and as shown in FIG. 1, the guanine (G) of the bulge base enters inside the base pair. It can also be outside the base pair as shown in FIG. In either case, there will be a space in the base pair due to the presence of the bulge base. In FIG. 1 and FIG. 2, such a space portion is indicated by a rectangle with hatching.
[0004]
As a method for detecting DNA having such a bulge base, a method utilizing the preferential binding of a DNA intercalator having a planar structure and capable of alkylating DNA to bulge is known. In the space formed by the presence of the bulge base shown in FIG. 1 or FIG. 2, the intercalation is performed using the stacking interaction between the aromatic ring and the base in the vicinity of the bulge.
However, such a method involves alkylation and intercalates when a space is generated, and does not act depending on the type of bulge base, and is due to the presence of a space that can be alkylated. Intercalation occurred and the bulge base itself was not judged.
Furthermore, many of the conventional methods using this method cause intercalation outside the DNA pair as shown in FIG. 3, and the bases present in the bulge cannot be distinguished.
[0005]
In addition, as a method for detecting DNA having a bulge base, a method utilizing the selective binding of a DNA repair protein such as MutS to a gene damage site is also known. This method is also specific to the type of bulge base. It was n’t.
The present inventors have established the concept of a bulge DNA recognition molecule capable of recognizing a bulge base, and have reported a 2-alkylcarbonylamino-1,8-naphthyridine derivative as a specific bulge DNA recognition molecule (JP-A-2001-2001). 89478). However, the bond between the bulge recognition molecule and the bulge base is extremely weak compared to the bond of a normal base pair, and a mismatch recognition molecule for a mismatch base pair separately developed by the present inventors (see JP-A-2001-149096). ) And the bond was weak. Therefore, even if the bulge recognition molecule is immobilized on a pup, it is difficult to immobilize DNA containing a bulge base.
[0006]
[Problems to be solved by the invention]
The present invention can also immobilize DNA containing a bulge base on a puppy by strengthening the binding between a bulge base in a double-stranded DNA strand in which a bulge base is present and a bulge recognition molecule. A bulge base recognition molecule is provided.
More specifically, an object of the present invention is to provide an improved bulge base-recognizing molecule that can bind to a bulge base as strongly as possible for immobilization. Furthermore, an object of the present invention is to provide a bulge recognition molecule that can be immobilized on a carrier such as a chip, and a method for detecting and identifying a bulge base using the bulge recognition molecule.
[0007]
[Means for Solving the Problems]
In order to solve the above problems, the present inventors have used an intercalator that hydrogen bonds with a bulge base, and the hydrogen bond complex of the bulge base and the intercalator is stacked on a double-stranded DNA and is a stable complex. Has been found (Japanese Patent Laid-Open No. 2001-89478). However, these bulge recognition molecules are weakly bound to a bulge base, and it is difficult to immobilize DNA containing the bulge base on a carrier such as a chip. The present inventors have intensively studied to improve this point. As a result, the bulge recognition molecule can form a covalent bond with a hydrogen-bonded bulge base, whereby a DNA containing the bulge base and the bulge recognition molecule Has been found to be fully coupled.
[0008]
That is, the present invention relates to a bulge base recognition molecule that can form a hydrogen bond specifically 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 further has a functional group capable of forming a covalent bond with a bulge base. More specifically, the bulge recognition molecule has the following general formula (I),
[0009]
[Chemical 2]

[0010]
(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 or a nitrogen atom; 15 alkoxy groups, wherein one or more carbon atoms in the alkoxy group may be substituted with an oxygen atom or a nitrogen atom, or a mono- or dialkylamino group having 1 to 15 carbon atoms. A mono- or dialkylamino group in which one or more carbon atoms in the alkylamino group may be substituted with an oxygen atom or a nitrogen atom,
R ₂ is a C 1-20 alkyl group having a functional group capable of forming a covalent bond with a bulge base as a substituent, and one or more carbon atoms in the alkyl group are an oxygen atom or a nitrogen atom The alkyl group which may be substituted by is shown. )
It is related with the bulge base recognition molecule | numerator which is a compound represented by these. The bulge recognition molecule of the present invention can further have a linker group for binding it to a carrier such as a chip. The present invention also relates to a bulge recognition molecule immobilized on a carrier by such a linker group.
[0011]
The present invention also provides a composition for recognizing a bulge base comprising the bulge base recognition molecule of the present invention as described above, and detects and identifies a bulge base in DNA using the bulge base recognition molecule of the present invention as described above. Alternatively, the present invention relates to a method for quantification and a measurement kit therefor.
Furthermore, the present invention relates to DNA in which a bulge base is stabilized by forming a hydrogen bond with a specific bulge base and stacking it on a base pair existing in the vicinity of the bulge base.
[0012]
The present inventors have developed a 1,8-naphthyridine derivative as a bulge recognition molecule for guanine (G) (Japanese Patent Laid-Open No. 2001-89478), and forms a covalent bond with a bulge base based on this molecule. Various methods have been studied. As a result, it was found that a covalent bond can be formed with a valid base by providing a reactive functional group at an appropriate distance from the bulge base recognition site of the bulge recognition molecule. As a bulge recognition molecule that can be covalently bonded to the bulge base of the present invention, for example, the following formula (II) derived from a 1,8-naphthyridine derivative,
[0013]
[Chemical 3]

[0014]
The present invention will be described based on 2- (β- (N-epoxymethyl-amino) -propionylamino) -7-methyl-1,8-naphthyridine represented by the following formula.
As shown in FIG. 4, the bulge recognition molecule of the present invention comprises a bulge base recognition part (in this example, a guanine recognition part) and a bulge base modification part (in this example, a guanine modification part). As shown, the bulge base recognition unit first recognizes the bulge base, and the bulge base recognition unit and the bulge base form a hydrogen bond. Next, it is considered that the active hydrogen in the side chain bends in the direction of the bulge base based on the polar part of the bulge base, and as a result, the reactive functional group of the bulge recognition molecule and the bulge base cause a chemical reaction.
[0015]
This was confirmed using the following oligomer (1) and oligomer (2).
*
Oligomer (1) 5′-ATTGCCGCTGA-3 ′
Oligomer (2) 3′-TAACG GACT-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. .
This oligomer (1), oligomer (2) and 2- (β- (N-epoxymethyl-amino) -propionylamino) -7-methyl-1,8-naphthyridine (hereinafter referred to as the present specification) of the formula (II) Was called “epoxynaphthyridine”) in a cacodylate buffer solution at room temperature. FIG. 6 shows a chart of the results of high performance liquid chromatography (HPLC) before the reaction and HPLC after 20 hours at room temperature (dT is used as an internal standard).
Before the reaction (upper chart in FIG. 6), peaks of internal standard dT, oligomer (2), oligomer (1), and epoxynaphthyridine were observed from the left side of the chart. In the chart, from the left side of the chart, an internal standard dT, a large peak of oligomer (2), a small peak of oligomer (1), and a large peak of a new product on the right side of each peak of epoxynaphthyridine were observed. This new product peak was confirmed to be a reaction product of the oligomer (1) and epoxynaphthyridine since the peak of the oligomer (1) was small.
As a result, it was found that the reaction rate between the oligomer (1) and epoxy naphthyridine was about 97%, and the reaction rate with the oligomer (2) was about 3%.
[0016]
According to the bulge recognition molecule of the present invention, the bulge recognition molecule and the DNA containing the bulge base are firmly bound to each other by forming a covalent bond with the bulge base. It is possible to selectively immobilize only the DNA containing, and to detect, identify or quantify it as DNA immobilized using a carrier such as a chip. For example, when testing whether a gene lacks one or several bases, prepare a fragment of the gene containing the missing part as a test gene fragment, and use the normal base sequence And then reacting in the presence of the bulge recognition molecule of the present invention, it is possible to detect, identify or quantify whether or not the analyte gene fragment has a base deficiency.
[0017]
Therefore, the bulge base recognition molecule of the present invention can be used as a bulge base recognition agent, and can be used as a bulge base recognition composition by combining with an appropriate carrier or measurement material.
The present invention also provides a measurement method for detecting, identifying or quantifying bulge bases in DNA using the bulge base recognition molecules or labeled or immobilized bulge base recognition molecules of the present invention. is there. The measurement method of the present invention may be the HPLC measurement described above, but the bulge recognition molecule of the present invention is immobilized on a chip or the like, and any of the DNAs to be assayed is labeled on a carrier such as the chip. The remaining label can be measured, but is not limited to these measurement methods. The gene fragment used for the measurement is not particularly limited, but preferably has a length of about 10 to 100 bases, more preferably about 10 to 50 bases and about 10 to 30 bases.
Furthermore, mismatch recognition molecules developed by the present inventors (see Japanese Patent Application Laid-Open No. 2001-149096) can also be used in such a bulge base assay. By using such a mismatch recognition molecule in combination, it is possible to test for the presence and type of a missing base in a sample gene fragment, and at the same time, also test for a change in base such as SNP.
[0018]
In addition, the bulge base recognition molecule of the present invention can be used alone, but by introducing a radioactive element at an appropriate position in the molecule or introducing a molecular species that emits chemiluminescence or fluorescence, etc. It can also be used after labeling. Furthermore, the bulge base recognition molecule of the present invention can be used by immobilizing it by binding directly or using a linker or the like to a polymer material such as polystyrene or a chip at an appropriate position. When immobilized and used, a bulge recognition molecule capable of selectively determining the bulge base of guanine (G), a bulge recognition molecule capable of selectively determining the bulge base of cytosine (C), thymine By arranging a bulge recognition molecule that can selectively determine the bulge base of (T) and a bulge recognition molecule that can selectively determine the bulge base of adenine (A), four kinds of bases are arranged. It is also possible to determine the presence of a bulge by a single test.
[0019]
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.
[0020]
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.
[0021]
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).
[0022]
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.
[0023]
By using the bulge base recognition molecule of the present invention, in DNA having one or more bulge bases, a hydrogen bond is formed with the bulge base by a specific base to stabilize it, and further shared with the bulge base. By forming a bond, DNA containing a bulge base can be stabilized. In particular, the bulge base recognition molecule of the present invention not only forms a hydrogen bond with a specific bulge base and forms a covalent bond, but also is stacked in the vicinity, preferably adjacent base pairs, and a bulge base is present. Nevertheless, relatively stable DNA can be obtained.
Therefore, according to the present invention, a bulge base is stabilized by forming a hydrogen bond with a specific bulge base and forming a covalent bond with the bulge base recognition molecule, and stacking on a base pair in the vicinity of the bulge base. The present invention provides a DNA containing a modified bulge base.
The DNA of the present invention has the bulge base recognition of the present invention in which the bulge base forms a “pair” similar to the base pair by hydrogen bonding with the bulge base recognition molecule of the present invention and forms a “pair” with the bulge base. The molecules are considered to be sandwiched and stacked in the vicinity, preferably a base forming an adjacent base pair.
[0024]
By using the bulge DNA recognition molecule of the present invention, a bulge DNA recognition molecule that cannot be achieved by conventional techniques can be immobilized with high sensitivity, so that it can be rapidly detected, identified or quantified in large quantities. Since the presence or absence of a bulge base or only a specific bulge base can be selected, detected, identified or quantified, the bulge recognition molecule of the present invention is useful for the treatment, prevention or diagnosis of various diseases associated with DNA damage.
Further, since the DNA of the present invention can exist relatively stably in a state having a bulge base, stabilization of DNA containing the bulge base, elucidation of the cause of bulge base generation and the repair mechanism of the bulge base, etc. It is also important as a research material.
[0025]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.
[0026]
Example 1 (Production Method of Epoxy Naphthyridine)
The reaction formula in the production of epoxy naphthyridine is shown below.
[0027]
[Formula 4]

[0028]
(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 the solvent was distilled off concentrated, the residue was purified by silica gel chromatography and purified by _{(CHCl 3 / MeOH = 10 1} ), adduct of interest (95.4mg, 0.29mmol, 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 = 8.8Hz), 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).
[0029]
(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 = 8.8Hz), 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)
[0030]
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′-ATTGCCGCTGA-3 ′
Oligomer (2) 3′-TAACG GACT-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. .
A cacodylate buffer solution (50 mM, pH 7.) containing d (TCAG GCAAT) / (ATTGCCGCTGA) (50 μM, standard concentration) obtained by hybridizing the oligomer (1) and oligomer (2) and dT (100 μM) as an internal standard. 0) was added eboxynaphthyridine (500 μM) produced in Example 1 and allowed to react at room temperature. 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.
The result of HPLC is shown in FIG.
[0031]
【The invention's effect】
The bulge recognition molecule of the present invention forms a hydrogen bond with a specific bulge base and not only forms a covalent bond, but also stably stacks with a nearby base pair, and strongly binds to a specific bulge base through a covalent bond. Therefore, it is possible to immobilize DNA containing a bulge base using the bulge recognition molecule of the present invention. Then, by immobilizing the bulge recognition molecule of the present invention on a carrier such as a chip, it becomes possible to selectively immobilize only DNA containing a specific bulge base on the carrier. Therefore, it is possible to rapidly detect, identify or quantify the types of bases with high sensitivity and in large quantities, which is extremely useful for the treatment, prevention or diagnosis of various diseases associated with DNA damage.
[Brief description of the drawings]
FIG. 1 schematically shows a bulge base (guanine in the figure) facing inward of a base pair.
FIG. 2 schematically shows a bulge base (guanine in the figure) facing outward from the base pair.
FIG. 3 schematically shows a state where intercalation is performed from the outside of a base pair to a bulge base (guanine in the figure) facing outward from the base pair.
[Fig. 4] Fig. 4 is a description based on the compounds illustrating the constituent parts of the bulge recognition molecule of the present invention.
FIG. 5 shows that the bulge recognition molecule of the present invention recognizes a bulge base and forms a hydrogen bond with the bulge base, and then the bulge base modification part of the bulge recognition molecule reacts with the bulge base to form a covalent bond. This is an example of modeling the state of formation.
FIG. 6 shows a HPLC chart before the reaction (upper chart in FIG. 6) and after the reaction (lower chart in FIG. 6) of the epoxy naphthyridine of the present invention and DNA containing a bulge base. Before the reaction (upper chart in FIG. 6), the internal standard dT, oligomer (2), oligomer (1), and epoxynaphthyridine peaks are shown from the left side of the chart, and after the reaction (lower chart in FIG. 6). From the left side of the chart, the peaks of internal standard dT, oligomer (2), oligomer (1), epoxynaphthyridine, and new product are shown.

Claims (11)

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 or a nitrogen atom; 15 alkoxy groups, wherein one or more carbon atoms in the alkoxy group may be substituted with an oxygen atom or a nitrogen atom, or a mono- or dialkylamino group having 1 to 15 carbon atoms. A mono- or dialkylamino group in which one or more carbon atoms in the alkylamino group may be substituted with an oxygen atom or a nitrogen atom,
R ₂ is an alkyl group having 3 to 7 carbon atoms having an epoxy group, aziridinyl group, or cyclopropyl group as a substituent, and one or more carbon atoms in the alkyl group are oxygen atoms or nitrogen atoms The alkyl group which may be substituted is shown. )
A bulge base recognition molecule in which the bulge base is guanine, which is a compound represented by: The bulge base recognition molecule according to claim 1, wherein the substituent of R ₂ is an epoxy group.   The bulge base recognition molecule according to claim 2, wherein the bulge base recognition molecule is 2- (β- (N-epoxymethyl-amino) -propionylamino) -7-methyl-1,8-naphthyridine.   The alkylene group is further provided as a linker group for fixing a carrier even if one or more carbon atoms are substituted by a nitrogen atom, an oxygen atom, a sulfur atom or an amide bond. Bulge base recognition molecule.   The bulge base recognition molecule according to claim 4, which is bound to a carrier.   The bulge base recognition molecule according to claim 6, wherein the carrier is a chip.   A bulge base recognition composition comprising the bulge base recognition molecule according to any one of claims 1 to 6, wherein the bulge base is guanine. A method for detecting, identifying or quantifying bulge base, which is guanine in DNA, using the bulge base recognition molecule according to any one of claims 1 to 6 (excluding application to a human body) .   The DNA of the subject is hybridized to DNA having a normal base sequence, and the hybridized DNA is immobilized in the presence of the bulge base recognition molecule according to any one of claims 1 to 6. 9. The method according to 8.   The method according to claim 9, wherein either DNA comprising a normal base sequence or DNA of a subject is labeled.   A DNA containing a bulge base that is stabilized guanine, wherein the bulge base recognition molecule according to any one of claims 1 to 6 is bound to the bulge base.

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