JP4011937B2 - Mismatch detection molecule - Google Patents

Description

【００１１】
（式中、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、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、窒素原子又はカルボニル基で置換されてもよいアルキル基を示す。）
で表される化合物又は当該化合物がプレートや機器分析用の検出装置などに固定化され得る化学構造に修飾された固定化物に関する。
【００１２】
さらに、本発明は、検体となる１本鎖のＤＮＡ又はＲＮＡと、それに対応する正常な塩基配列を有するＤＮＡ又はＲＮＡとをハイブリダイズさせ、次いで当該ハイブリダイズしたＤＮＡ又はＲＮＡにおける正常な塩基対を形成することができない塩基の対を次の一般式（Ｉ）、
Ａ−Ｌ−Ｘ （Ｉ）
（式中、Ａは正常な塩基対を形成することができない塩基の対の片方の塩基と対を形成し得る化学構造部分、Ｘは前記Ａの構造部分と対を形成する塩基と正常な塩基対を形成すべき塩基とは異なる塩基と対を形成し得る化学構造部分、Ｌは化学構造部分Ａ及びＸを結合するリンカー構造を示す。）
で表される塩基対のミスマッチ認識分子であって、特定の塩基と対を形成し得る化学構造部分Ａ及び前記Ａの構造部分と対を形成する塩基と正常な塩基対を形成すべき塩基とは異なる塩基と対を形成し得る化学構造部分Ｘ、並びに当該化学構造部分Ａ及びＸを結合するリンカー部分Ｌを有することを特徴とする化合物を用いて、当該正常な塩基対を形成することができない塩基の対に擬似的に塩基対を形成させ、当該擬似的な塩基対の状態を測定することからなる正常な塩基対を形成することができない塩基の対を検出、同定することからなるＤＮＡ又はＲＮＡにおける塩基配列の異常を検出する方法に関する。
なお、以下の説明においては、前記した「正常な塩基対を形成することができない塩基の対の片方の塩基と対を形成し得る化学構造部分（一般式（Ｉ）におけるＡ及びＸの部分）」のことを単に「塩基認識部位」ということもある。
【００１３】
本発明者らは、２本鎖ＤＮＡ中に生成する不対塩基（バルジ塩基）を持つＤＮＡ（バルジＤＮＡ）に特異的に結合し、安定化する分子であるバルジＤＮＡ認識分子を開発してきた（特開２００１−８９４７８号）。このバルジ認識分子は、バルジ塩基の存在により生じてくる空間に、芳香環とバルジ近傍の塩基とのスタッキング相互作用を利用してインターカーレーションし、安定化されているものであるが、本発明者らはこのようなバルジ認識分子を２分子、リンカーのような結合鎖で結合させることにより各々のバルジ認識分子が、塩基対のミスマッチ部分においてバルジ塩基と同様な塩基対を形成し、しかもこれらのバルジ認識分子の両者が２本鎖を形成しているＤＮＡやＲＮＡの鎖の中に比較的安定に取り込まれることを見出してきた（特開２００１−１４９０９６号）。このような比較的大きな分子種がＤＮＡやＲＮＡの鎖の中に比較的安定に取り込まれることは驚くべきことであり、かつこのような特性を利用することにより、ハイブリダイズしている核酸の中で塩基対がミスマッチを生じている箇所を簡便に特定し得ることを見出し、これをミスマッチ認識分子として示してきた。
【００１４】
しかし、このものは塩基に対する特異性が強く、特定のミスマッチにのみ強く取り込まれるものであった。そこで、本発明者らは塩基に対する結合が、正常な塩基対を形成するよりは弱いが、同定や検出のためには十分な対を形成し得る分子種の開発を行ってきたところ、このような分子種を製造することができることを見出した。例えば、次式（III）で示される、
【００１５】
【化５】

【００１６】
分子種の右側部分（一般式（Ｉ）におけるＸ部分に相当する部分）のナフチリドンはいずれの塩基とも比較的弱く結合し、いずれのミスマッチ塩基に対しても適度の結合能により取り込まれことを見出した。
前記式（III）の左側部分（一般式（Ｉ）におけるＡ部分に相当する部分）は、以前に報告してきたグアニンと水素結合を形成し、かつ周囲の塩基とスタッキング効果により安定化され得る２−置換−１，８−ナフチリジン構造であり、この構造がグアニン（Ｇ）と対を形成することは、既に報告してきたとおりである。
【００１７】
核酸の２本鎖中においてグアニンに対するミスマッチが存在している場合において、前記式（III）のミスマッチ認識分子が各々のミスマッチに対する取り込みついて検討した。
この検討のために、次の３種のオリゴマーを用いた。

オリゴマー（３）は正常な塩基対を有するものであり、オリゴマー（１）及び（２）は前記の塩基配列の上の＊印を示した部分においてミスマッチが生じているものである。
【００１８】
これらのオリゴマー（１）及び（２）、並びにオリゴマー（３）のそれぞれについて、前記式（III）のミスマッチ認識分子の存在下、又は非存在下における二本鎖ＤＮＡの融解温度（Ｔｍ）を調べた。
その結果を次の表１に示す。

【００１９】
表１は左から、試験をしたオリゴマーの塩基配列、式（III）のミスマッチ認識分子の非存在下での融解温度（Ｔｍ）（℃）、式（III）のミスマッチ認識分子の存在下での融解温度（Ｔｍ）（℃）、各融解温度（Ｔｍ）（℃）の差（ΔＴｍ）（℃）を示している。ここに、融解温度（Ｔｍ）の差（ΔＴｍ）は、
ΔＴｍ＝（式（III）存在下でのＴｍ）−（式（III）非存在下でのＴｍ）
から算出されたものである。
この結果からも明らかなように、ミスマッチが存在していた場合にはΔＴｍの値が大きく相違することになり、ミスマッチの存在を確認することができる。
そして、ミスマッチの種類によりΔＴｍの値が相違してくるだけでなく、式（III）のミスマッチ認識分子の非存在下での融解温度（Ｔｍ）も相違している。さらに本発明で重要なことは、式（III）のミスマッチ認識分子が存在した場合における融解温度（Ｔｍ）の相違である。例えば、オリゴマー（１）の場合にはこのＴｍの値は４０．４℃であり、この値は正常なオリゴマー（３）の値４０．５℃とほぼ同じ値になっているのに対して、オリゴマー（２）のこのＴｍの値は３６．１℃極めて低い温度になっている。これは式（III）の分子種における右側部分（一般式（Ｉ）におけるＸ部分に相当する部分）に対する各塩基との結合能が反映された結果である。即ち、式（III）の分子種の場合にはアデニン（Ａ）塩基とはほぼ正常な対に近い結合能を有するが（オリゴマー（１）の場合）、グアニン（Ｇ）塩基とは結合はするが比較的弱い結合能しか有さない（オリゴマー（２）の場合）結果が反映されたものであり、このことにより、本発明のミスマッチ認識分子を用いることにより、調べようとする遺伝子にミスマッチ（例えば、ＳＮＰなど）が存在していることが判明するだけではなく、当該ミスマッチにおける相手側の塩基を知ることが可能となることがわかった。
【００２０】
本発明のミスマッチ認識分子の基本的なことについては前記した特開２００１−１４９０９６号に記載されているが、本発明のミスマッチ認識分子は、一方の塩基を認識する部分を塩基の種類に対して非特異的にし、かつ正常な塩基対に対しては影響を与えない分子種であることを特徴とするものである。
したがって、本発明のミスマッチ認識分子の塩基認識部位（一般式（Ｉ）におけるＡ及びＸの化学構造部分）において、Ａ部分は塩基の種類に対して特異性が強い構造を有する部分であり、Ｘ部分は塩基の種類に対して比較的非特異的な結合ができることを特徴とするものである。
【００２１】
このように、本発明は、２個の異なる塩基認識部位を適当な長さでかつ適当な自由度を有するリンカーで結合させた化合物が、２本鎖の核酸における塩基対のミスマッチ部分において、一方は特異的にかつ安定に対を形成し、他方は比較的に非特異的に結合するという特徴により、簡便な方法でミスマッチの存在を判定することができるだけでなく、ミスマッチの種類も判定することができるという基本的なミスマッチ判定法を提供するものであり、前記に例示したグアニン（Ｇ）に対するミスマッチに限定されるものではない。
前記した例では、グアニン（Ｇ）に対するミスマッチを例に取り、グアニン塩基と安全な水素結合を形成する１，８−ナフチリジン誘導体を一方の塩基認識部位に用いたミスマッチ認識分子を示したが、ミスマッチの認識はグアニン（Ｇ）に対するミスマッチに限定されるものではない。本発明にミスマッチ認識分子における塩基認識部位は、ミスマッチの塩基の片方を認識し当該塩基とワトソン−クリック（Watson-Crick）型の塩基対を形成することができ、周囲の塩基によるスタッキング効果を得られる分子種を選択することにより、例示したグアニンに限らず、各種の塩基と塩基対を形成し得ることができる構造を使用することができる。例えば、ミスマッチの塩基がシトシンの場合には、塩基認識部位として２−アミノナフチリジン−４−オン又はその誘導体などが、ミスマッチの塩基がアデニンの場合には、２−キノロン誘導体、例えば３−（２−アミノエチル）−２−キノロン又はその誘導体などが、また、ミスマッチの塩基がチミンの場合には、２−アミノナフチリジン−７−オン又はその誘導体などが用いられる。
【００２２】
特定のミスマッチの塩基に特異的に認識される本発明のミスマッチ認識分子における塩基認識部位は、水素結合を形成するための水素結合部位と、近傍の塩基にスタッキングされるための平面構造を有している複素環式芳香族基を有するものが好ましい。
【００２３】
また、本発明の一般式（Ｉ）で表される化合物におけるリンカー部Ｌとしては、２個の塩基認識部位を適当な長さで適当な自由度を与えるものであれ特に制限されるものではないが、例えば、炭素数１〜２０、好ましくは１〜１５、より好ましくは１〜１２の直鎖状又は分枝状の飽和又は不飽和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、窒素原子又はカルボニル基で置換されてもよいアルキル基が挙げられる。好ましいリンカーとしては、前記した式（III）の化合物のように両端がアミド結合の部分を有し、中央部に窒素原子を有するものが挙げられる。
このリンカー部分は、２個の塩基認識部位を結合させるだけでなく、このリンカー部分から担体に固定化するための枝を結合させることもできる。例えば、リンカー中央部付近の窒素原子の箇所からさらに末端に担体と結合するための官能基などを有するアルキレン基のような枝を延ばして、必要に応じて担体に固定化することもできる。
【００２４】
本発明の一般式（Ｉ）における塩基認識部位Ａ又はＸとリンカー部Ｌとの結合は、炭素−炭素結合であってもよいが合成の簡便さから官能基による結合が好ましい。官能基による結合としては、エーテル結合、エステル結合、アミド結合、リン酸による結合など種々のタイプのものを選択することができるが、アミド結合が好ましい。
本発明のミスマッチ塩基認識分子における、グアニン（Ｇ）に対するミスマッチに好ましい一般式（Ｉ）の化合物として、次の一般式（II）、
【００２５】
【化６】

【００２６】
（式中、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、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、窒素原子又はカルボニル基で置換されてもよいアルキル基を示す。）
で表される化合物又はその固定化物が挙げられる。ここにおける「固定化物」とは、前記した化合物が担体に固定化されている状態のもの又は固定化され得るように前記した「枝」をのばした状態の化合物をいう。
本発明の一般式（II）における置換基Ｒ_１としては、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。
また、Ｒ_２におけるアルキル基は前記したアルキル基であって、一般式（II）に示されているように２価のアルキル基である。
【００２７】
また、本発明のミスマッチ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、例えばリンカー部分やリンカーから固定化のためなどのための延ばされた枝などに、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。なお、測定手段としての標識化は、検出対象のＤＮＡやＲＮＡなどの核酸部分の標識化によることもできる。
さらに、本発明のミスマッチ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と直接又はアルキレン基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。
【００２８】
本発明のミスマッチ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、前記した１，８−ナフチリジン誘導体は、２−アミノ−１，８−ナフチリジン又は２−アミノ−７−メチル−１，８−ナフチリジンをＮ−保護−４−アミノ−酪酸の反応性誘導体、例えば酸塩化物を反応させて、２位のアミノ基をアシル化した後、アミノ基を保護基を脱保護して製造することができる。この際の保護基としては、塩酸塩やアシル基やアルコキシカルボニル基などのペプチド合成において使用されるアミノ保護基を使用することができる。
このようにして得られた塩基認識部位を、両末端にカルボキシル基又はその反応性誘導体基を有するリンカー用の化合物と反応させることにより目的のミスマッチ塩基認識分子を得ることができる。この際に、リンカー用化合物の分子中に窒素原子などの反応性の基が存在している場合には、前記した保護基などで適宜保護して使用することができる。
【００２９】
本発明のミスマッチ塩基認識分子は、これをミスマッチの塩基の対の検出するための試薬又は検出剤として使用することができ、また、適当な担体と組み合わせることによりミスマッチの塩基の対を検出するための組成物とすることができる。また、正常な塩基対を形成することができない塩基の対に擬似的に塩基対を形成させるための塩基対生成剤として使用することもできる。ここに、「擬似的な塩基対」というのは、天然に存在する塩基の対とは異なる塩基の対であるという意味であり、塩基対の強度を意味するものではない。なお、本明細書において使用される「正常な塩基対」とは天然に存在する塩基の対であって、Ｇ−Ｃ、Ａ−Ｔ、又はＡ−Ｕの塩基対をいう。
本発明は、さらに前記した本発明のミスマッチ塩基認識分子、及び検出、同定用の資材、例えば化学発光や蛍光のための試薬や緩衝液などの資材からなる、正常な塩基対を形成することができない塩基の対に擬似的に塩基対を形成させ、当該擬似的な塩基対の形成を測定することからなる正常な塩基対を形成することができない塩基の対を検出、同定するためのキットを提供するものである。
さらに、本発明は、本発明のミスマッチ塩基認識分子又は標識化若しくは固定化された本発明のミスマッチ塩基認識分子を使用して、ＤＮＡ中のミスマッチしている塩基の対を検出、同定又は定量するための方法を提供するものである。
【００３０】
本発明のミスマッチ認識分子の塩基認識部位を用いることにより、例えばクロスハイブリダイゼーション法において、調べようとしている遺伝子又はその断片と正常な遺伝子又はその断片における１個又は２個以上のミスマッチの塩基の対（例えば、遺伝子における一塩基変異（ＳＮＰ）など）を簡便に、その有無及びミスマッチにおけるミスマッチ塩基の種類を判定することができる。例えば、式（III）に示される本発明のミスマッチ認識分子を用いて、調べようとする遺伝子の断片、例えば１０〜１００塩基、好ましくは１０〜５０塩基、１０〜３０塩基程度の断片とし、これに対応する正常な塩基配列を有する遺伝子の断片を用意し、正常な遺伝子の断片と調べようとする遺伝子の断片を、式（III）に示される本発明のミスマッチ認識分子の存在下及び非存在下のそれぞれにおいてハイブリダイズさせて、それらの融解温度（Ｔｍ）を測定することによりミスマッチの有無（この例ではＳＮＰなどの有無）や、ミスマッチの塩基の種類を判定することができることになる。この場合に一般式（Ｉ）におけるＡ構造の部分は特定の塩基、例えばグアニン（Ｇ）と対を形成することになることから、この例の場合ではミスマッチは正常な塩基配列を有する遺伝子の断片のグアニン（Ｇ）塩基の箇所で起きていることも判明することになる。
【００３１】
また、本発明のミスマッチ認識分子は、塩基認識部位（一般式（Ｉ）におけるＡ構造の部分）がミスマッチの特定の塩基と安定な対を形成し、当該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ミスマッチの塩基の対が安定化されたミスマッチ塩基対を含有するＤＮＡを提供するものである。
本発明のこのＤＮＡは、ミスマッチの塩基の対が本発明のミスマッチ認識分子の塩基認識部位と水素結合により塩基対と同様な「対」（擬似的な塩基対）を形成し、かつミスマッチの塩基と「対」を形成している本発明のミスマッチ認識分子の塩基認識部位が近傍、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安定なＤＮＡを提供するものである。
【００３２】
本発明のミスマッチ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ミスマッチの塩基の対を高感度でかつ簡便に検出、同定又は定量することが出来、ミスマッチの塩基の対に特異的でかつ安定なＤＮＡを形成することから、ＤＮＡ損傷に伴う各種疾患の治療、予防又は診断に応用することが可能である。
また、本発明のＤＮＡは、ミスマッチの塩基の対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、ミスマッチの塩基の対を含有するＤＮＡの安定化や、ミスマッチの発生原因やミスマッチの修復機構の解明などの研究材料として利用することも可能である。
【００３３】
前記した新たな対（本発明のミスマッチ認識分子とミスマッチの塩基との対）を測定するための手段としては、融解温度（Ｔｍ）を指標とすることができるが、これに限定されるものではなく、例えば化学発光や蛍光、放射性同位体などの標識化によっても行うことができる。本発明のミスマッチ認識分子は低分子有機化合物であり、新たな対を形成した場合にはこの分子が核酸中に取り込まれるので、未反応の本発明のミスマッチ認識分子と核酸類の両者を比較的簡便に分離することができる。
また、前記したように本発明のミスマッチ認識分子を担体に固定化して使用することもできる。例えば、本発明の各種のミスマッチに特異的なミスマッチ認識分子をタイタープレートなどのプレートに固定化し、これに前記の２本鎖の核酸、好ましくは標識化された核酸を加え、数分間インキュベートした後、核酸類を除去すると本発明のミスマッチ認識分子と特異的に反応した核酸は固定化された本発明のミスマッチ認識分子にトラップされ、標識により検出、同定することができることになる。
【００３４】
また、本発明のミスマッチ認識分子を表面プラズモン共鳴（ＳＰＲ）の検出用チップの金属薄膜上に固定化することも可能である。このＳＰＲによる場合には、前記の２本鎖の核酸を含有する試料液を検出用チップの表面に流すだけで、ミスマッチの有無を特異的に検出することが可能となる。
さらに、他の多くの検出手段に本発明のミスマッチ認識分子を応用することも可能であり、本発明はこれらの特定の検出手段に限定されるものではない。
【００３５】
【実施例】
次に、具体的な試験例により本発明を詳細に説明するが、本発明はこれらの具体例に限定されるものではない。
実施例１（式（III）の化合物の合成）
次式に示す化学反応に従って標記の化合物を合成した。
【００３６】
【化７】

【００３７】
（ＤＭＦはジメチルホルムアミドを示し、Ｅｔはエチル基を示し、Ａｃはアセチル基を示す。）
（１）Ｎ−ｔ−ブトキシカルボニルイミノ−３，３’−ビス（ペンタフルオロフェニルプロピオネート）（N-(tert-Butoxycarbonyl)imino-3,3’-bis(pentafluorophenyl propionate) ）(1.5 g, 2.53mmol)のＤＭＦ(5 mL)溶液に２−アミノ−７−メチル−１，８−ナフチリジン(180 mg, 1.14 mmol) とジイソプロピルエチルアミン(163 mg, 1.26 mmol)を加え、室温で１５時間撹拌した。溶媒を留去した後、残差をシリカゲルカラムクロマトグラフィーで精製して、モノナフチリジン置換体(1.29 g, 90%)を得た:
^１Ｈ ＮＭＲ（ＣＤＣｌ_３，４００ＭＨｚ） δ：
9.01 (br, 1 H), 8.44 (d, 1 H, J = 8.8 Hz),
8.12 (d, 1 H, J = 8.8 Hz), 7.99 (d, 1 H, J = 8.0 Hz),
7.26 (d, 1 H, J = 8.0 Hz), 3.66 (m, 4 H), 2.90 (m, 2 H),
2.74 (m, 2 H), 2.73 (s, 3 H), 1.42 (s, 9 H);
ＦＡＢＭＳ（ＮＢＡ）、ｍ／ｅ ： ５６９［（Ｍ＋Ｈ）^＋， ］，
ＨＲＭＳ ：
計算値 Ｃ_２６Ｈ_２６Ｏ_５Ｎ_４Ｆ_５ ［（Ｍ＋Ｈ）^＋］ ５６９．１８２１，
実測値 ５６９．１８２７
【００３８】
（２）前記（１）で得たノナフチリジン置換体(300 mg, 0.53 mmol) のＤＭＡ(2 mL)溶液に７−アミノメチル−（２Ｈ）ヒドロ−８−アザキノリン−２−オン（7-(aminomethyl)hydro-8-azaquinolin-2-one）(93 mg, 0.53 mmol)とジイソプロピルエチルアミン(77 mg, 0.6 mmol)を加え、室温で１５時間撹拌した。溶媒を留去した後、残差をシリカゲルカラムクロマトグラフィーで精製して、ナフチリジン−ナフチリドン−Ｎ−Ｂｏｃ（ｔ−ブトキシカルボニル基）保護体(227 mg,
77%) を得た。
^１Ｈ ＮＭＲ（ＣＤＣｌ_３，４００ＭＨｚ） δ：
11.29 (br, 1 H), 9.08 (br, 1H), 8.38 (d, 1 H, J = 8.8 Hz),
8.06 (d, 1 H, J = 8.0 Hz), 7.95 (d, 1 H, J = 8.0 Hz),
7.81 (d, 1 H, J = 8.0 Hz), 7.64 (d, 1 H, J = 9.6 Hz),
7.27 (d, 1 H, J = 8.0 Hz), 7.22 (d, 1 H, J = 8.0 Hz),
6.64 (d, 1 H, J = 9.6 Hz), 4.63 (d, 2 H, J = 6.0 Hz),
3.58 (t, 2 H, J = 6.8 Hz), 3.57 (t, 2 H, J = 6.8 Hz),
2.71 (t, 2H, J = 6.8 Hz), 2.70 (s, 1 H),
2.54 (t, 2 H, J = 6.8 Hz), 1.36 (s, 9 H) ;
ＦＡＢＭＳ （ＮＢＡ），ｍ／ｅ： ５６０ ［（Ｍ＋Ｈ）＋］；
ＨＲＭＳ ；
計算値 Ｃ_２９Ｈ_３４Ｏ_５Ｎ_７ ［（Ｍ＋Ｈ）^＋］ ５６０．２６２１，
実測値 ５６０．２６１８
【００３９】
（３）前記（２）で得たＢｏｃ保護体 (62 mg, 0.11 mmol)のＣＨＣｌ_３（３ｍＬ）溶液に、４ＭＨＣｌ（２ｍＬ）の酢酸エチル溶液を加え、室温で３０分撹拌した。溶媒を留去目的物（収率定量的）を得た。
^１Ｈ ＮＭＲ（ＣＤＣｌ_３，４００ＭＨｚ） δ：
11.45 (br, 1 H), 8.67 (t, 1 H, J = 5.6 Hz),
8.31 (d, 1 H, J = 8.8 Hz), 7.98 (d, 1 H, J = 8.8 Hz),
7.88 (d, 1 H, J = 8.0 Hz), 7.65 (d, 1 H, J = 7.6 Hz),
7.53 (d, 1 H, J = 9.6 Hz), 7.15 (d, 1 H, J = 7.6 Hz),
7.14 (t, 1 H, J = 8.0 Hz), 6.58 (d, 1 H, J = 9.6 Hz),
4.65 (d, 2 H, J = 5.6 Hz), 3.08 (t, 2 H, J = 6.0 Hz),
3.06 (t, 2 H, J = 6.0 Hz), 2.65 (t, 2 H, J = 6.0 Hz),
2.58 (t, 2 H, J = 6.0 Hz), 2.57 (s, 3 H).
【００４０】
実施例２ （ミスマッチＤＮＡの融解温度（Ｔｍ）の測定）
次の（１）〜（３）の３種の１１塩基からなるヌクレオチドオリゴマーの各々１００μＭを、１００ｍＭのＮａＣｌ及び式（III）の化合物２００μＭを含む１０ｍＭカコジル酸ナトリウム緩衝液（ｐＨ７．０）に添加して、実施例１で製造した一般式（III）のミスマッチ認識分子の存在下及び非存在下における融解温度（Ｔｍ）をそれぞれ測定した。

オリゴマー（３）は正常な塩基対を有するものであり、オリゴマー（１）及び（２）は前記の塩基配列の上の＊印を示した部分においてミスマッチが生じているものである。
結果を表１に示す。
【００４１】
【発明の効果】
本発明の一般式（Ｉ）で示されるミスマッチ認識分子を用いることにより、当該ミスマッチ認識分子の一方の側（一般式（Ｉ）のＡ構造の部分）において特定のミスマッチ塩基を認識し、他方の側（一般式（Ｉ）のＸ構造の部分）においてミスマッチのいずれの種類の塩基とも塩基の種類に応じて結合することができることから、融解温度（Ｔｍ）を測定することによりミスマッチの有無及びミスマッチの塩基種類を簡便な手段により、迅速に、且つ確実に判定することができる。 本発明のミスマッチ認識分子を使用することにより、遺伝子の断片におけるＳＮＰの存在の有無や種類の判定を迅速にかつ大量に処理することが可能となる。[0001]
BACKGROUND OF THE INVENTION
The present invention allows a base pair that cannot form a normal base pair to form a pseudo base pair in a base pair that cannot form a normal base pair, and A method for detecting and identifying a base pair that cannot form a normal base pair consisting of measuring a state, a reagent therefor, a kit containing the same, a compound thereof, and a DNA or RNA using the method The present invention relates to a method for detecting an abnormality in a base sequence.
[0002]
[Prior art]
When nucleic acids such as DNA and RNA are hybridized to form a double strand, a base to be paired is determined. For example, guanine (G) has cytosine (C) and adenine (A) has thymine (T). Usually, all the bases form such a pair and hybridize, but sometimes such a pair cannot be formed in a part of the base sequence.
For example, most bases can form such a pair when subjected to conditions under which one DNA can hybridize with another DNA, but one or several partial bases May not be able to form a correct pair. Such a base pair that cannot form a normal base pair is hereinafter referred to as a mismatch.
[0003]
On the other hand, studies on various genetic diseases resulting from differences in one or more bases have been conducted recently. For example, there is a genetic disease in which one base has a gene (SNP (Single Nucleotide Polymorphism)) different from a normal one, and elucidation of the genetic disease has been attracting attention. When such a gene is hybridized with a normal gene, most bases can form normal base pairs and thus hybridize, but mismatches occur for one base pair. become.
At present, a method for detecting such a mismatch is generally a method for comparing the hybridization efficiency of double-stranded DNA. However, in order to use this method, it is necessary to know in advance the base sequence of a DNA containing a mismatch, which requires a lot of labor, and is not suitable as a method for processing many samples. In addition, there is a technique that utilizes the selective binding of DNA repair proteins such as MutS to sites of gene damage. However, when using proteins, thermal stability and active structure ( (Folding) must be maintained, and there are many restrictions on usage conditions, and the operation is complicated, making it difficult to efficiently detect mismatches.
[0004]
Thus, it is very difficult to detect some mismatches in hybridized DNA and the like, and its sensitivity is insufficient, and establishment of a method that can detect this easily and with high sensitivity is required. Yes.
By the way, the present inventors have developed a bulge DNA recognition molecule that is a molecule that specifically binds and stabilizes DNA (bulge DNA) having an unpaired base (bulge base) generated in double-stranded DNA. (Japanese Patent Laid-Open No. 2001-89478). This bulge recognition molecule not only forms hydrogen bonds with unpaired bases, but also intercalates into the space created by the presence of bulge bases using the stacking interaction between the aromatic ring and bases near the bulges, It has been stabilized.
The inventors have further studied the action against unpaired bases using the stacking effect due to the presence of such peripheral bases. As a result, even in locations where base pair mismatches have occurred, It has been found that a compound having two molecular species capable of forming a pair can be incorporated relatively stably by such a stacking effect. Specifically, a bis (2-methylnaphthyridine) amide derivative has a GG mismatch or a GA mismatch. It has been shown that it can be detected (Japanese Patent Laid-Open No. 2001-149096). However, this can be incorporated very sensitively and stably into a specific mismatch, but is insufficiently incorporated into other mismatches, i.e., it is too specific for a particular mismatch. It was not practical. For example, although it has high specificity for a GG mismatch, sufficient uptake has not necessarily been made for a GA mismatch or a GT mismatch.
[0005]
As a method for examining gene mutation, there is a method in which an oligomer DNA of about 50 bases of a gene to be examined for the presence of mutation and a wild type gene without the mutation is mixed, heated and cooled to cross-hypridize the two genes. If there is a mutation in the gene to be examined, an abnormality is found in the melting temperature or melting temperature difference of the gene. Although this operation is relatively simple, it is only known that there is a mutation, and it is impossible to know what mutation is occurring at which position.
According to the method reported by the present inventor described above (Japanese Patent Laid-Open No. 2001-149096), it is possible to know the presence of a specific mismatch by this cross-hybridization method, but this mismatch recognition molecule has specificity. For example, even when examining mismatches to guanine bases, it is necessary to prepare a plurality of mismatch recognition molecules such as a GG mismatch, a GA mismatch, and a GT mismatch.
[0006]
[Problems to be solved by the invention]
The present invention provides a method capable of detecting a base pair mismatch with respect to a specific base in a simpler and more sensitive manner in such a high-pridization method.
The present invention also provides a method capable of easily detecting a mismatch present in a double-stranded DNA strand with high sensitivity and a detection kit therefor.
[0007]
[Means for Solving the Problems]
As a result of further investigation on mismatch recognition molecules, the present inventors have found that not all mismatch recognition molecules that are specifically incorporated into specific mismatches, but all mismatches for a certain base, for example, not only GG mismatches for guanine bases, The present inventors have found a mismatch recognition molecule that can be incorporated into any of GG mismatch, GA mismatch, and GT mismatch and that can detect the degree of incorporation.
The present invention relates to a base pair that cannot form a normal base pair,
The following general formula (I),
A-L-X (I)
(Wherein, A is a chemical structure part that can form a pair with one base of a base pair that cannot form a normal base pair, and X is a normal base and a base that forms a pair with the structural part of A) (A chemical structure moiety capable of forming a pair with a base different from the base to be paired, and L represents a linker structure that connects the chemical structures A and X.)
And the normal base using a compound having a chemical structural part A and a chemical structural part X that can form a pair with each of the bases, and a linker part L that connects the chemical structural parts A and X. Detecting a base pair that cannot form a normal base pair by forming a pseudo base pair in a base pair that cannot form a pair and measuring the formation of the pseudo base pair, It relates to a method of identification.
[0008]
The present invention also provides a normal base pair comprising: forming a pseudo base pair in a base pair that cannot form a normal base pair, and measuring the formation of the pseudo base pair. In the method for detecting and identifying a base pair that cannot be formed, the following general formula (I) for forming a pseudo base pair in a base pair that cannot form a normal base pair,
A-L-X (I)
(Wherein, A is a chemical structure part that can form a pair with one base of a base pair that cannot form a normal base pair, and X is a normal base and a base that forms a pair with the structural part of A) (A chemical structure moiety capable of forming a pair with a base different from the base to be paired, and L represents a linker structure that connects the chemical structures A and X.)
It is related with the reagent for making a base pair which forms the base pair which cannot form a normal base pair to form a pseudo base pair.
In the present invention, a pseudo base pair is formed in a base pair that cannot form a normal base pair consisting of the above-described reagent of the present invention and materials for detection and identification. The present invention relates to a kit for detecting and identifying a base pair that cannot form a normal base pair consisting of measuring formation.
[0009]
Further, the present invention provides the following general formula (II),
[0010]
[Formula 4]

[0011]
(Wherein 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, An alkoxy group in which 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, Represents a mono- or dialkylamino group in which one or more carbon atoms in the alkylamino group may be substituted with oxygen or nitrogen atoms;
R ₂ Is an alkyl group having 1 to 20 carbon atoms, and one or more carbon atoms in the alkyl group may be substituted with an oxygen atom, a nitrogen atom or a carbonyl group. )
Or an immobilized product in which the compound is modified to have a chemical structure that can be immobilized on a plate, a detection device for instrumental analysis, or the like.
[0012]
Furthermore, the present invention hybridizes a single-stranded DNA or RNA serving as a specimen with a corresponding DNA or RNA having a normal base sequence, and then normal base pairs in the hybridized DNA or RNA are determined. A base pair that cannot be formed is represented by the following general formula (I):
A-L-X (I)
(Wherein, A is a chemical structure part that can form a pair with one base of a base pair that cannot form a normal base pair, and X is a normal base and a base that forms a pair with the structural part of A) (A chemical structure moiety capable of forming a pair with a base different from the base to be paired, and L represents a linker structure that connects the chemical structures A and X.)
A base pair mismatch recognition molecule represented by the formula: a chemical structural part A capable of forming a pair with a specific base, and a base which forms a normal base pair with a base which forms a pair with the structural part of A Can form the normal base pair by using a compound having a chemical structural part X capable of pairing with different bases and a linker part L connecting the chemical structural parts A and X. DNA consisting of detecting and identifying a base pair that cannot form a normal base pair by forming a pseudo base pair in an incapable base pair and measuring the state of the pseudo base pair Alternatively, the present invention relates to a method for detecting a base sequence abnormality in RNA.
In the following description, the above-described “chemical structure part capable of forming a pair with one base of a base pair that cannot form a normal base pair (parts A and X in the general formula (I))” "Is sometimes simply referred to as" base recognition site ".
[0013]
The present inventors have developed a bulge DNA recognition molecule that is a molecule that specifically binds and stabilizes DNA (bulge DNA) having an unpaired base (bulge base) generated in double-stranded DNA ( JP-A-2001-89478). This bulge recognition molecule is stabilized by intercalating into the space generated by the presence of the bulge base by utilizing the stacking interaction between the aromatic ring and the base in the vicinity of the bulge. By connecting two such bulge recognition molecules with a linking chain such as a linker, each bulge recognition molecule forms a base pair similar to a bulge base at the mismatched portion of the base pair. It has been found that both of these bulge recognition molecules are relatively stably incorporated into DNA or RNA strands forming double strands (Japanese Patent Laid-Open No. 2001-149096). It is surprising that such relatively large molecular species are relatively stably incorporated into DNA and RNA strands, and by utilizing such properties, it is possible to And found that it is possible to easily identify the location where the base pair is mismatched, and this has been shown as a mismatch recognition molecule.
[0014]
However, this was highly specific for the base and was strongly incorporated only into specific mismatches. Therefore, the present inventors have developed a molecular species capable of forming a sufficient pair for identification and detection, although the binding to the base is weaker than that of forming a normal base pair. It has been found that various molecular species can be produced. For example, represented by the following formula (III):
[0015]
[Chemical formula 5]

[0016]
It has been found that naphthyridone in the right part of the molecular species (the part corresponding to the X part in the general formula (I)) binds to any base relatively weakly and is incorporated into any mismatched base with moderate binding ability. It was.
The left side part of the formula (III) (the part corresponding to the A part in the general formula (I)) forms a hydrogen bond with guanine reported previously and can be stabilized by the stacking effect with the surrounding base 2 -It is a substituted-1,8-naphthyridine structure, and this structure forms a pair with guanine (G) as already reported.
[0017]
When mismatches to guanine exist in the double strand of the nucleic acid, the mismatch recognition molecule of the formula (III) was examined for incorporation into each mismatch.
For this study, the following three oligomers were used.

The oligomer (3) has a normal base pair, and the oligomers (1) and (2) are those in which a mismatch has occurred in the portion indicated by * in the base sequence.
[0018]
For each of these oligomers (1) and (2) and oligomer (3), the melting temperature (Tm) of the double-stranded DNA in the presence or absence of the mismatch recognition molecule of formula (III) is examined. It was.
The results are shown in Table 1 below.

[0019]
Table 1 shows, from the left, the base sequence of the tested oligomer, the melting temperature (Tm) (° C.) in the absence of the mismatch recognition molecule of formula (III), and the presence of the mismatch recognition molecule of formula (III). The melting temperature (Tm) (° C.) and the difference (ΔTm) (° C.) between the melting temperatures (Tm) (° C.) are shown. Here, the difference (ΔTm) in the melting temperature (Tm) is
ΔTm = (Tm in the presence of formula (III)) − (Tm in the absence of formula (III))
It is calculated from
As is apparent from this result, when there is a mismatch, the value of ΔTm greatly differs, and the presence of the mismatch can be confirmed.
In addition to the difference in ΔTm depending on the type of mismatch, the melting temperature (Tm) in the absence of the mismatch recognition molecule of formula (III) also differs. What is more important in the present invention is the difference in melting temperature (Tm) when the mismatch recognition molecule of formula (III) is present. For example, in the case of the oligomer (1), this Tm value is 40.4 ° C., which is almost the same value as the normal oligomer (3) value 40.5 ° C. This Tm value of the oligomer (2) is extremely low at 36.1 ° C. This is a result of reflecting the binding ability with each base to the right side portion (the portion corresponding to the X portion in the general formula (I)) in the molecular species of the formula (III). That is, in the case of the molecular species of the formula (III), it has a binding ability close to a normal pair with the adenine (A) base (in the case of the oligomer (1)), but binds with the guanine (G) base. Has a relatively weak binding ability (in the case of the oligomer (2)), which reflects the result, and by using the mismatch recognition molecule of the present invention, a mismatch ( For example, it has been found that not only the existence of SNP etc.) is present, but also the counterpart base in the mismatch can be known.
[0020]
The basics of the mismatch recognition molecule of the present invention are described in the aforementioned Japanese Patent Application Laid-Open No. 2001-149096, but the mismatch recognition molecule of the present invention has a portion that recognizes one base relative to the type of base. The molecular species is non-specific and does not affect normal base pairs.
Accordingly, in the base recognition site of the mismatch recognition molecule of the present invention (the chemical structure portion of A and X in the general formula (I)), the A portion is a portion having a structure having a strong specificity for the type of base, The moiety is characterized by relatively non-specific binding to the type of base.
[0021]
Thus, in the present invention, a compound in which two different base recognition sites are linked with a linker having an appropriate length and an appropriate degree of freedom is obtained in a mismatched part of a base pair in a double-stranded nucleic acid. The ability to form specific and stable pairs, while the other binds relatively non-specifically, not only can the presence of mismatches be determined in a simple way, but also the type of mismatch Is provided, and is not limited to the above-described mismatch for guanine (G).
In the above example, a mismatch recognition molecule using a 1,8-naphthyridine derivative that forms a safe hydrogen bond with a guanine base as one base recognition site was shown by taking a mismatch to guanine (G) as an example. Is not limited to a mismatch to guanine (G). The base recognition site in the mismatch recognition molecule according to the present invention can recognize one of the mismatched bases and form a Watson-Crick type base pair with the base, thereby obtaining a stacking effect by surrounding bases. By selecting the molecular species to be used, not only the exemplified guanine but also a structure capable of forming base pairs with various bases can be used. For example, when the mismatched base is cytosine, 2-aminonaphthyridin-4-one or a derivative thereof as a base recognition site, and when the mismatched base is adenine, a 2-quinolone derivative such as 3- (2 -Aminoethyl) -2-quinolone or a derivative thereof, and when the mismatched base is thymine, 2-aminonaphthyridin-7-one or a derivative thereof is used.
[0022]
The base recognition site in the mismatch recognition molecule of the present invention that is specifically recognized by a specific mismatch base has a hydrogen bonding site for forming a hydrogen bond and a planar structure for stacking to a neighboring base. Those having a heterocyclic aromatic group are preferred.
[0023]
In addition, the linker portion L in the compound represented by the general formula (I) of the present invention is not particularly limited as long as it gives an appropriate degree of freedom with two base recognition sites having an appropriate length. Is, for example, a linear or branched saturated or unsaturated alkyl group having 1 to 20 carbon atoms, preferably 1 to 15 carbon atoms, more preferably 1 to 12 carbon atoms, Examples include alkyl groups in which further carbon atoms may be substituted with oxygen atoms, nitrogen atoms or carbonyl groups. Preferred linkers include those having both ends having an amide bond and a nitrogen atom at the center, such as the compound of formula (III) described above.
This linker moiety can bind not only two base recognition sites but also a branch for immobilization on the carrier from this linker moiety. For example, a branch such as an alkylene group having a functional group for binding to the carrier at the terminal can be further extended from the position of the nitrogen atom in the vicinity of the central portion of the linker, and can be immobilized on the carrier as necessary.
[0024]
The bond between the base recognition site A or X and the linker part L in the general formula (I) of the present invention may be a carbon-carbon bond, but is preferably a bond by a functional group for the convenience of synthesis. As the bond by the functional group, various types such as an ether bond, an ester bond, an amide bond, and a bond by phosphoric acid can be selected, but an amide bond is preferable.
As a compound of the general formula (I) preferred for mismatch to guanine (G) in the mismatch base recognition molecule of the present invention, the following general formula (II),
[0025]
[Chemical 6]

[0026]
(Wherein 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, An alkoxy group in which 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, Represents a mono- or dialkylamino group in which one or more carbon atoms in the alkylamino group may be substituted with oxygen or nitrogen atoms;
R ₂ Is an alkyl group having 1 to 20 carbon atoms, and one or more carbon atoms in the alkyl group may be substituted with an oxygen atom, a nitrogen atom or a carbonyl group. )
Or a compound immobilized thereon. The “immobilized product” herein refers to a compound in a state where the above-described compound is immobilized on a carrier or a compound in which the above-mentioned “branch” is extended so that the compound can be immobilized.
Substituent R in the general formula (II) of the present invention ₁ As, 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 to 10 carbon atoms. Mono- or di-substituted by an alkoxy group consisting of 7 linear or branched alkyl groups, 1 to 15 carbon atoms, preferably 1 to 10 and more preferably 1 to 7 linear or branched alkyl groups And mono- or dialkylamino groups that are used. 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.
R ₂ The alkyl group in is an alkyl group as described above, and is a divalent alkyl group as shown in the general formula (II).
[0027]
In addition, the mismatch base recognition molecule of the present invention can be used alone, but at an appropriate position in the molecule, for example, a linker part or a branch extended for immobilization from the linker. In addition, it can be used after being labeled by introducing a radioactive element or introducing a molecular species that emits chemiluminescence or fluorescence. Note that the labeling as a measuring means can be performed by labeling a nucleic acid portion such as DNA or RNA to be detected.
Furthermore, the mismatch base recognition molecule of the present invention can be used by immobilizing it with a polymer material such as polystyrene directly or using an alkylene group or the like at an appropriate position.
[0028]
The mismatch base recognizing molecule of the present invention is a low molecular weight organic compound and can be appropriately produced by a general organic synthesis method. For example, the aforementioned 1,8-naphthyridine derivative is a reactive derivative of 2-amino-1,8-naphthyridine or 2-amino-7-methyl-1,8-naphthyridine as N-protected-4-amino-butyric acid, For example, after reacting an acid chloride to acylate the amino group at the 2-position, the amino group can be produced by deprotecting the protecting group. As the protecting group in this case, an amino protecting group used in peptide synthesis such as hydrochloride, acyl group or alkoxycarbonyl group can be used.
The target mismatch base recognition molecule can be obtained by reacting the base recognition site thus obtained with a linker compound having a carboxyl group or a reactive derivative group at both ends. In this case, when a reactive group such as a nitrogen atom is present in the molecule of the linker compound, it can be used by appropriately protecting with the above-described protecting group.
[0029]
The mismatched base recognition molecule of the present invention can be used as a reagent or detection agent for detecting a mismatched base pair, and for detecting a mismatched base pair in combination with an appropriate carrier. It can be set as the composition of this. Moreover, it can also be used as a base pair production | generation agent for making a base pair which cannot form a normal base pair artificially form a base pair. Here, the “pseudo base pair” means a base pair different from a naturally occurring base pair, and does not mean the strength of the base pair. The “normal base pair” used in the present specification is a naturally occurring base pair and refers to a GC, AT, or AU base pair.
The present invention further forms a normal base pair consisting of the above-described mismatched base recognition molecule of the present invention and materials for detection and identification, such as reagents and buffers for chemiluminescence and fluorescence. A kit for detecting and identifying a base pair that cannot form a normal base pair, by forming a pseudo base pair in an incapable base pair and measuring the formation of the pseudo base pair. It is to provide.
Furthermore, the present invention uses the mismatched base recognition molecule of the present invention or the labeled or immobilized mismatched base recognition molecule of the present invention to detect, identify or quantify mismatched base pairs in DNA. It provides a method for
[0030]
By using the base recognition site of the mismatch recognition molecule of the present invention, for example, in the cross-hybridization method, the gene or fragment thereof to be examined and one or more mismatched base pairs in the normal gene or fragment thereof The presence / absence of a single base mutation (SNP, etc. in a gene, for example) and the type of mismatch base in the mismatch can be determined. For example, using the mismatch recognition molecule of the present invention represented by the formula (III), a fragment of the gene to be examined, for example, a fragment of about 10 to 100 bases, preferably about 10 to 50 bases and about 10 to 30 bases, A fragment of a gene having a normal base sequence corresponding to is prepared, and a normal gene fragment and a gene fragment to be examined are detected in the presence and absence of the mismatch recognition molecule of the present invention represented by the formula (III) By hybridizing each of them below and measuring their melting temperature (Tm), it is possible to determine the presence or absence of mismatch (in this example, the presence or absence of SNP) and the type of mismatched base. In this case, the portion of the A structure in the general formula (I) forms a pair with a specific base, for example, guanine (G). In this case, the mismatch is a fragment of a gene having a normal base sequence. It is also found that this occurs at the guanine (G) base.
[0031]
In the mismatch recognition molecule of the present invention, the base recognition site (part of the A structure in the general formula (I)) forms a stable pair with a specific base of mismatch and is stacked on a base pair existing in the vicinity of the base. Thus, a DNA containing a mismatched base pair in which a mismatched base pair is stabilized is provided.
In the DNA of the present invention, the mismatched base pair forms a “pair” (pseudo-base pair) similar to the base pair by hydrogen bonding with the base recognition site of the mismatch recognition molecule of the present invention, and the mismatched base. Provides a stable DNA that is stacked sandwiched between the bases of the mismatch recognition molecules of the present invention that form a “pair” with the bases that are adjacent, preferably adjacent base pairs. To do.
[0032]
By using the mismatch recognition molecule of the present invention, mismatched base pairs that cannot be achieved by conventional techniques can be detected, identified or quantified with high sensitivity and easily, and specific and stable to mismatched base pairs. Therefore, it can be applied to 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 mismatched base pair, the DNA containing the mismatched base pair is stabilized, the cause of the mismatch or the occurrence of the mismatch It can also be used as research material for elucidating the repair mechanism.
[0033]
As a means for measuring the above-described new pair (a pair of the mismatch recognition molecule of the present invention and a mismatched base), the melting temperature (Tm) can be used as an index, but is not limited thereto. For example, it can also be performed by labeling chemiluminescence, fluorescence, radioisotope, or the like. The mismatch recognition molecule of the present invention is a low molecular weight organic compound, and when a new pair is formed, this molecule is incorporated into the nucleic acid, so that both the unreacted mismatch recognition molecule of the present invention and the nucleic acids are relatively separated. It can be easily separated.
Further, as described above, the mismatch recognition molecule of the present invention can be used by immobilizing it on a carrier. For example, after mismatch recognition molecules specific for various mismatches of the present invention are immobilized on a plate such as a titer plate, the above-mentioned double-stranded nucleic acid, preferably labeled nucleic acid, is added thereto, and incubated for several minutes. When the nucleic acids are removed, the nucleic acid specifically reacted with the mismatch recognition molecule of the present invention is trapped by the immobilized mismatch recognition molecule of the present invention, and can be detected and identified by the label.
[0034]
It is also possible to immobilize the mismatch recognition molecule of the present invention on a metal thin film of a surface plasmon resonance (SPR) detection chip. In the case of this SPR, it is possible to specifically detect the presence or absence of mismatch only by flowing the sample solution containing the double-stranded nucleic acid on the surface of the detection chip.
Furthermore, the mismatch recognition molecule of the present invention can be applied to many other detection means, and the present invention is not limited to these specific detection means.
[0035]
【Example】
Next, the present invention will be described in detail with specific test examples, but the present invention is not limited to these specific examples.
Example 1 (Synthesis of compound of formula (III))
The title compound was synthesized according to the chemical reaction shown in the following formula.
[0036]
[Chemical 7]

[0037]
(DMF represents dimethylformamide, Et represents an ethyl group, and Ac represents an acetyl group.)
(1) N- (tert-Butoxycarbonyl) imino-3,3′-bis (pentafluorophenyl propionate) (1.5 g, 2.53 2-amino-7-methyl-1,8-naphthyridine (180 mg, 1.14 mmol) and diisopropylethylamine (163 mg, 1.26 mmol) were added to a DMF (5 mL) solution of mmol), and the mixture was stirred at room temperature for 15 hours. After distilling off the solvent, the residue was purified by silica gel column chromatography to obtain a mononaphthyridine substituted product (1.29 g, 90%):
¹ 1 H NMR (CDCl ₃ , 400 MHz) δ:
9.01 (br, 1 H), 8.44 (d, 1 H, J = 8.8 Hz),
8.12 (d, 1 H, J = 8.8 Hz), 7.99 (d, 1 H, J = 8.0 Hz),
7.26 (d, 1 H, J = 8.0 Hz), 3.66 (m, 4 H), 2.90 (m, 2 H),
2.74 (m, 2 H), 2.73 (s, 3 H), 1.42 (s, 9 H);
FABMS (NBA), m / e: 569 [(M + H) ⁺ ,],
HRMS:
Calculated value C ₂₆ H ₂₆ O ₅ N ₄ F ₅ [(M + H) ⁺ ] 569.1821,
Measured value 569.1828
[0038]
(2) 7-Aminomethyl- (2H) hydro-8-azaquinolin-2-one (7- (7- (7H)) was added to the DMA solution (2 mL) of the nonnaphthyridine substituted product (300 mg, 0.53 mmol) obtained in (1) above. Aminomethyl) hydro-8-azaquinolin-2-one) (93 mg, 0.53 mmol) and diisopropylethylamine (77 mg, 0.6 mmol) were added, and the mixture was stirred at room temperature for 15 hours. After the solvent was distilled off, the residue was purified by silica gel column chromatography to obtain a protected naphthyridine-naphthylidone-N-Boc (t-butoxycarbonyl group) (227 mg,
77%).
¹ 1 H NMR (CDCl ₃ , 400 MHz) δ:
11.29 (br, 1 H), 9.08 (br, 1H), 8.38 (d, 1 H, J = 8.8 Hz),
8.06 (d, 1 H, J = 8.0 Hz), 7.95 (d, 1 H, J = 8.0 Hz),
7.81 (d, 1 H, J = 8.0 Hz), 7.64 (d, 1 H, J = 9.6 Hz),
7.27 (d, 1 H, J = 8.0 Hz), 7.22 (d, 1 H, J = 8.0 Hz),
6.64 (d, 1 H, J = 9.6 Hz), 4.63 (d, 2 H, J = 6.0 Hz),
3.58 (t, 2 H, J = 6.8 Hz), 3.57 (t, 2 H, J = 6.8 Hz),
2.71 (t, 2H, J = 6.8 Hz), 2.70 (s, 1 H),
2.54 (t, 2 H, J = 6.8 Hz), 1.36 (s, 9 H);
FABMS (NBA), m / e: 560 [(M + H) +];
HRMS;
Calculated value C ₂₉ H ₃₄ O ₅ N ₇ [(M + H) ⁺ ] 560.2621
Actual value 560.2618
[0039]
(3) CHCl of Boc protector (62 mg, 0.11 mmol) obtained in (2) above ₃ To the (3 mL) solution was added 4M HCl (2 mL) in ethyl acetate and stirred at room temperature for 30 minutes. The solvent was distilled off to obtain the desired product (yield quantitative).
¹ 1 H NMR (CDCl ₃ , 400 MHz) δ:
11.45 (br, 1 H), 8.67 (t, 1 H, J = 5.6 Hz),
8.31 (d, 1 H, J = 8.8 Hz), 7.98 (d, 1 H, J = 8.8 Hz),
7.88 (d, 1 H, J = 8.0 Hz), 7.65 (d, 1 H, J = 7.6 Hz),
7.53 (d, 1 H, J = 9.6 Hz), 7.15 (d, 1 H, J = 7.6 Hz),
7.14 (t, 1 H, J = 8.0 Hz), 6.58 (d, 1 H, J = 9.6 Hz),
4.65 (d, 2 H, J = 5.6 Hz), 3.08 (t, 2 H, J = 6.0 Hz),
3.06 (t, 2 H, J = 6.0 Hz), 2.65 (t, 2 H, J = 6.0 Hz),
2.58 (t, 2 H, J = 6.0 Hz), 2.57 (s, 3 H).
[0040]
Example 2 (Measurement of melting temperature (Tm) of mismatched DNA)
100 μM of each of the following three types of nucleotide oligomers consisting of 11 bases (1) to (3) is added to 10 mM sodium cacodylate buffer (pH 7.0) containing 100 mM NaCl and 200 μM of the compound of formula (III). Then, the melting temperature (Tm) in the presence and absence of the mismatch recognition molecule of the general formula (III) produced in Example 1 was measured.

The oligomer (3) has a normal base pair, and the oligomers (1) and (2) are those in which a mismatch has occurred in the portion indicated by * in the base sequence.
The results are shown in Table 1.
[0041]
【The invention's effect】
By using the mismatch recognition molecule represented by the general formula (I) of the present invention, a specific mismatch base is recognized on one side of the mismatch recognition molecule (part of the A structure of the general formula (I)), and the other Since it can bind to any type of mismatched base (depending on the type of base) on the side (X structure portion of the general formula (I)), the presence or absence of mismatch and mismatch are determined by measuring the melting temperature (Tm). The base type can be determined quickly and reliably by a simple means. By using the mismatch recognition molecule of the present invention, the presence / absence and type of SNP in a gene fragment can be determined quickly and in large quantities.

Claims (10)

A pseudo base pair is formed between the compound represented by the following general formula (I) and a base that cannot form a normal base pair, and the state of the pseudo base pair is measured. A method for detecting an abnormality in a base sequence in DNA or RNA, comprising detecting and identifying a base pair that cannot form a normal base pair.
A-L-X (I)
Wherein A is 2-R ₁ -1,8-naphthyridine, 2-aminonaphthyridine-4-one, 3- (2-aminoethyl) -2-quinolone or 2-aminonaphthyridin-7-one; X is a (1-hydro-2-oxo-1,8-naphthyridin-7-yl) -methyl-amino-carbonyl group , R ₁ is a hydrogen atom, an alkyl group having 1 to 15 carbon atoms, An alkyl group in which one or more carbon atoms in the alkyl group may be substituted with an oxygen atom or a nitrogen atom, an alkoxy group having 1 to 15 carbon atoms, and one or more carbons in the alkoxy group An alkoxy group in which the atom may be substituted with an oxygen atom or a nitrogen atom, or a mono- or dialkylamino group having 1 to 15 carbon atoms, wherein one or more carbon atoms in the alkylamino group are oxygen atoms Indicates a nitrogen atom optionally substituted mono- or dialkylamino group which may be, L is one or more of the carbon atoms is an oxygen atom, a nitrogen atom or an alkyl of carbon atoms which may be have 20 substituted with a carbonyl group The linker structure which couple | bonds A and X consisting of group is shown .).   The method according to claim 1, wherein the bond between A and X and the linker moiety L in the compound represented by the general formula (I) is a carboxylic acid amide bond.   The method according to claim 1 or 2, wherein the compound represented by the general formula (I) is immobilized on a carrier.   The method according to any one of claims 1 to 3, wherein the compound represented by the general formula (I) is labeled. In the method according to any one of claims 1 to 4, the following general formula (I) for forming a pseudo base pair in a base pair that cannot form a normal base pair:
A-L-X (I)
Wherein A is 2-R ₁ -1,8-naphthyridine, 2-aminonaphthyridine-4-one, 3- (2-aminoethyl) -2-quinolone or 2-aminonaphthyridin-7-one; X is a (1-hydro-2-oxo-1,8-naphthyridin-7-yl) -methyl-amino-carbonyl group , R ₁ is a hydrogen atom, an alkyl group having 1 to 15 carbon atoms, An alkyl group in which one or more carbon atoms in the alkyl group may be substituted with an oxygen atom or a nitrogen atom, an alkoxy group having 1 to 15 carbon atoms, and one or more carbons in the alkoxy group An alkoxy group in which the atom may be substituted with an oxygen atom or a nitrogen atom, or a mono- or dialkylamino group having 1 to 15 carbon atoms, wherein one or more carbon atoms in the alkylamino group are oxygen atoms Indicates a nitrogen atom optionally substituted mono- or dialkylamino group which may be, L is one or more of the carbon atoms is an oxygen atom, a nitrogen atom or an alkyl of carbon atoms which may be have 20 substituted with a carbonyl group The linker structure which couple | bonds A and X which consists of group is shown .   The reagent according to claim 5, wherein the reagent is a base pair generating agent for causing a base pair that cannot form a normal base pair to form a pseudo base pair.   A pseudo base pair is formed on a base pair that cannot form a normal base pair, which is composed of the reagent according to claim 5 and materials for detection and identification, and the state of the pseudo base pair is determined. A kit for detecting and identifying a base pair that cannot be formed into a normal base pair consisting of measurement. The following general formula (II)

(In the formula, 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, An alkoxy group having 1 to 15 carbon atoms in which one or more carbon atoms in the alkoxy group may be substituted with an oxygen atom or a nitrogen atom, or a mono or dialkyl having 1 to 15 carbon atoms An amino 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 or a nitrogen atom;
R ₂ represents an alkyl group having 1 to 20 carbon atoms, and one or more carbon atoms in the alkyl group may be substituted with an oxygen atom, a nitrogen atom or a carbonyl group. )
Or a compound immobilized thereon. The compound represented by the general formula (II) is represented by the following general formula (III),

The compound according to claim 8, wherein A base incapable of hybridizing a single-stranded DNA or RNA serving as a specimen with a corresponding DNA or RNA having a normal base sequence and then forming a normal base pair in the hybridized DNA or RNA And the following general formula (I):
A-L-X (I)
Wherein A is 2- R ₁ -1,8-naphthyridine, 2-aminonaphthyridine-4-one, 3- (2-aminoethyl) -2-quinolone or 2-aminonaphthyridin-7-one; X is a (1-hydro-2-oxo-1,8-naphthyridin-7-yl) -methyl-amino-carbonyl group , R ₁ is a hydrogen atom, an alkyl group having 1 to 15 carbon atoms, An alkyl group in which one or more carbon atoms in the alkyl group may be substituted with an oxygen atom or a nitrogen atom, an alkoxy group having 1 to 15 carbon atoms, and one or more carbons in the alkoxy group An alkoxy group in which the atom may be substituted with an oxygen atom or a nitrogen atom, or a mono- or dialkylamino group having 1 to 15 carbon atoms, wherein one or more carbon atoms in the alkylamino group are oxygen atoms Or a mono- or dialkylamino group which may be substituted with a nitrogen atom, wherein L represents an alkyl having 1 to 20 carbon atoms in which one or more carbon atoms may be substituted with an oxygen atom, a nitrogen atom or a carbonyl group The linker structure which combines A and X consisting of a group is shown.
A pseudo base pair is formed with the compound represented by the above, and the state of the pseudo base pair is measured to detect and identify a base pair that cannot form a normal base pair. A method for detecting a base sequence abnormality in DNA or RNA.