JP2005289810A - Fluorescent reagent used for selectively detecting double-stranded nucleic acid and containing fluorescent dye group and intercalate group - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new fluorescent reagent. <P>SOLUTION: The fluorescent reagent containing a fluorescent dye group and an intercalate group can selectively detect a double-stranded DNA or a double-stranded RNA in a manner that when this reagent comes into contact with the double-stranded DNA or the double-stranded RNA, the intercalate group intercalates between base pairs in the double-stranded nucleic acid, and as a result, the quenching caused by stacking between the fluorescent dye group and the intercalate group in the reagent molecule is broken off so that the fluorescent intensity is elevated. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

（式中、Xはインターカレート基または蛍光色素基を表し、Yはα-アミノ酸を構成する2価の基を表し、Zはα-アミノ酸を構成する1価の基を表し、ｎは1から8の整数を表す。但し、式IにおけるそれぞれのXは、お互いに異なるもしくは同じ蛍光色素基またはインターカレート基であってもよく、式Iにおいては必ずインターカレート基および蛍光色素基の両者が存在する。また、式IにおけるそれぞれのYおよびZも、お互いに異なるものであっても同じものであってもよい）。
【０００６】
【発明の実施の形態】
本発明の蛍光試薬は、蛍光色素基およびインターカレート基を含有する二本鎖核酸を選択的に検出できる蛍光試薬であって、二本鎖核酸と接触することにより該蛍光試薬分子中のインターカレート基が二本鎖核酸の塩基対間にインターカレートして、該蛍光試薬分子中における蛍光色素基とインターカレート基とのスタッキングによる消光が解消して蛍光強度が増強し、それによって二本鎖核酸を選択的に検出することのできる蛍光試薬である。
本発明の蛍光試薬は、その分子内に蛍光色素基とインターカレート基とを有し、それらがスタッキングすることにより消光現象を起こしており、二本鎖核酸と接触することによって、インターカレート基が二本鎖核酸の塩基対間にインターカレートして、消光が解消し得る構造を有するものであれば、如何なるものであってもよい。このような本発明の蛍光試薬としては、例えば、反応性の官能基を有する適当なスペーサー鎖を介して蛍光色素基とインターカレート基を結合させた蛍光試薬などが挙げられる。
【０００７】
本発明の蛍光試薬としては、蛍光色素基で置換されたα−アミノ酸残基とインターカレート基で置換されたα−アミノ酸残基を含むα−アミノ酸残基からなるペプチドか好ましい。即ち、α−アミノ酸残基からなるペプチドに蛍光色素基とインターカレート基とを置換させたペプチドが好ましい。更には、蛍光色素基およびインターカレート基は、2から10個、更には2から5個、特に2個のα−アミノ酸残基おきに置換されているのが好ましい。即ち、蛍光色素基で置換されたα−アミノ酸残基およびインターカレート基で置換されたα−アミノ酸残基が、2から10個、更には2から5個、特に2個のα−アミノ酸残基おきに存在するのが好ましい。また、蛍光色素基で置換されたα-アミノ酸残基は、インターカレート基で置換されたα−アミノ酸残基の間に存在するのが好ましい。このような場合には、インターカレート基が、二本鎖核酸の塩基対間に効果的にインターカレートして、蛍光試薬分子中における蛍光色素基とインターカレート基とのスタッキングによる消光が効果的に解消して蛍光強度が著しく増強し、それによって二本鎖核酸を選択的に検出することのできる。また、本発明の蛍光試薬であるペプチドは、全α−アミノ酸残基数が、4から40個、特に4から25個であるペプチドが好ましい。更には全α−アミノ酸残基数4から16個、特に4から10個であるペプチドが好ましい。
また、本発明では、蛍光色素基は、インターカレーターとしての機能を有しない蛍光色素基が好ましい。
【０００８】
より具体的には、本発明の蛍光試薬は、下記式Ｉで表されるペプチド、そのN末端もしくはC末端が置換されたその誘導体またはそれらの塩が好ましい。
【化５】

（式中、Xはインターカレート基または蛍光色素基を表し、Yはα-アミノ酸を構成する2価の基を表し、Zはα-アミノ酸を構成する1価の基を表し、ｎは1から8の整数を表す。但し、式IにおけるそれぞれのXは、お互いに異なるもしくは同じ蛍光色素基またはインターカレート基であってもよく、式Iにおいては必ずインターカレート基および蛍光色素基の両者が存在する。また、式IにおけるそれぞれのYおよびZも、お互いに異なるものであっても同じものであってもよい）。
上記式Ｉで表されるペプチド、そのN末端もしくはC末端が置換されたその誘導体は新規な化合物である。
【０００９】
本発明の蛍光試薬におけるインターカレート基、並びに式ＩにおけるXのインターカレート基としては、二本鎖DNAもしくは二本鎖RNAの塩基対の間に平行して挿入し得るものであればいずれの基であってもよい。かかるインターカレート基としては、例えば、置換基を有していてもよいアクリジン骨格を有する基、置換基を有していてもよいアントラキノン骨格を有する基、置換基を有していてもよいアントラセン骨格を有する基、置換基を有していてもよいナフタレンイミド骨格を有する基、置換基を有していてもよいナフタレンジイミド骨格を有する基、置換基を有していてもよいピレン骨格を有する基などが挙げられる。ここで言う置換基としては、例えば、塩素、フッ素、臭素などのハロゲン原子；メチル、エチル、プロピル、ブチル、ペンチル、ヘキシル、トリフルオロメチルなどのハロゲン原子で置換されていてもよい炭素原子数1から6のアルキル基；メトキシ、エトキシ、ブトキシ、ペントキシ、ヘキソキシなどの炭素原子数1から6のアルコキシ基；ホルミル、アセチル、ブタノイル、ペンタノイル、ヘキサノイルなどの炭素原子数1から7のアシル基；アセチルオキシ、ブタノイルオキシ、ペンタノイルオキシ、ヘキサノイルオキシなどの炭素原子数2から7のアシルオキシ基；アミノメチル、アミノエチル、アミノプロピル、メチルアミノメチル、ジメチルアミメチル、ジメチルアミノプロピル、ジメチルアミノブチルなどの炭素原子数1から6のアルキル基が1つもしくは2つ置換されていてもよい炭素原子数1から6のアミノアルキル基などが挙げられる。これらの置換基は、アクリジン骨格、アントラキノン骨格、アントラセン骨格等のいずれの位置に置換されていてもよく、また2つ以上の複数の置換基がこれらの骨格に置換されていてもよい。
また、アクリジン骨格、アントラキノン骨格、アントラセン骨格等には、本発明の蛍光試薬、並びに式ＩのペプチドにおけるYと結合するための官能基を有していてもよく、このような官能基としては、例えば、-CO-、-NH-、-SO₂-、-PO₂-、-S-、-N(NH)NH-、これらの官能基が炭素原子数1から6のアルキレン基の末端もしくは側鎖に結合した基などが挙げられる。
【００１０】
上記した置換基を有していてもよいアクリジン骨格を有する基、置換基を有していてもよいアントラキノン骨格を有する基、置換基を有していてもよいアントラセン骨格を有する基、置換基を有していてもよいナフタレンイミド骨格を有する基、置換基を有していてもよいナフタレンジイミド骨格を有する基、置換基を有していてもよいピレン骨格を有する基の具体的な例として以下のものが挙げられる。
【００１１】
置換基を有していてもよいアクリジン骨格を有する基
【化６】

置換基を有していてもよいアントラキノン骨格を有する基
【化７】

置換基を有していてもよいアントラセン骨格を有する基
【化８】

置換基を有していてもよいナフタレンイミド骨格を有する基
【化９】

置換基を有していてもよいナフタレンジイミド骨格を有する基
【化１０】

置換基を有していてもよいピレン骨格を有する基
【化１１】

【００１２】
本発明の蛍光試薬における蛍光色素基、並びに式ＩにおけるXの蛍光色素基としては、蛍光を発する基であれば特に限定されず、いずれの基であってもよい。本発明では、蛍光色素基は、インターカレーターとしての機能を有しない蛍光色素基が好ましい。このような蛍光色素基としては、例えば、置換基を有していてもよいキサンテン骨格を有する基、置換基を有していてもよいインドール骨格を有する基、置換基を有していてもよい含ホウ素および窒素インダセン骨格を有する基などが挙げられる。ここで言う置換基としては、上記したインターカレート基の場合の置換基と同様のものが挙げられる。更には、カルボキシ基、スルフォン酸基、イミノ基、あるいはこれらの官能基が炭素原子数1から6のアルキレン基の末端もしくは側鎖に結合した基などが挙げられる。また、キサンテン骨格を有する基の場合には、キノリジジン環、ピロール環などが縮合する形態の置換基が挙げられる。インドール骨格を有する基の場合には、2つのインドール骨格が炭素原子数1から6のアルキレン基、アルケニレン基などを介して結合したものでもよい。含ホウ素および窒素インダセン骨格を有する基の場合には、フェニル基、あるいは上記したインターカレート基の場合の置換基と同様の置換基が置換したフェニル基などが置換していてもよい。
また、上記したインターカレート基の場合と同様に、本発明の蛍光試薬、並びに式ＩのペプチドにおけるYと結合するための官能基を有していてもよい。
このような蛍光色素基としては、具体的には、以下のようなものが好ましいものとして挙げられる。
【００１３】
置換基を有していてもよいキサンテン骨格を有する基
【化１２】

置換基を有していてもよいインドール骨格を有する基
【化１３】

置換基を有していてもよい含ホウ素および窒素インダセン骨格を有する基
【化１４】

【００１４】
式Ｉにおいて、それぞれのXは、お互いに異なるもしくは同じ蛍光色素基またはインターカレート基であってもよく、式Iにおいては必ずインターカレート基および蛍光色素基の両者が存在する。特に、式Ｉのペプチドにおいては、蛍光色素基とインターカレート基とはお互いに隣接して存在するのが好ましく、特に蛍光色素基の両側にインターカレート基が存在するような構造のペプチドが好ましい。式Ｉにおいて、Yはα-アミノ酸を構成する2価の基（結合手を2つ持つ）を表し、Zはα−アミノ酸を構成する1価の基（結合手を1つ持つ）を表す。即ち、Yはα−アミノ酸から-CH(NH₂)COOHを除いた残りの該α−アミノ酸を構成する2価の基を表し、Zは１価の基を表す。具体的には、Yは-A-W-で表される基であり、ここで、Aはメチレン、エチレン、プロピレン、メチルメチレンンなどの炭素原子数1から6の直鎖もしくは分岐アルキレン基を表し、Wは単結合、NH、CO、CONH、NHCO、O、SまたはHNC(NH)NHを表す。ZはA-W-Hで表される基である。これらのYおよびZは、α−アミノ酸である、例えばアラニン、リシン、ノルリシン、アスパラギン酸、グルタミン、セリン、システインなどから-CH(NH₂)COOHを除いた残りの該α−アミノ酸を構成するそれぞれ2価の基あるいは1価の基を表す。式IにおけるそれぞれのYおよびZは、お互いに異なるものであっても同じものであってもよい。式Ｉのペプチドにおいて、ｎは1から8の整数を表す。好ましくは、nは1から5、特に好ましくは、nは1から3の整数である。
【００１５】
本発明の蛍光試薬であるペプチド並びに式Ｉのペプチドは、そのN末端もしくはC末端が置換されたその誘導体またはそれらの塩であってもよい。N末端が置換された誘導体としては、例えば、ホルミル、アセチル、ブタノイル、ペンタノイル、ヘキサノイルなどの炭素原子数1から7のアシル基；メトキシカルボニル、ブトキシカルボニル、t-ブトキシカルボニル、ペンチルオキシカルボニル、ヘキシルオキシカルボニルなどの炭素原子数2から7のアルコキシカルボニル基等がN末端のアミノ基に置換された誘導体などが挙げられる。C末端が置換された誘導体としては、メチル、エチル、プロピル、ブチル、ペンチル、ヘキシル、トリフルオロメチルなどのハロゲン原子で置換されていてもよい炭素原子数1から6のアルキル基；アミノメチル、アミノエチル、アミノプロピル、メチルアミノメチル、ジメチルアミノメチル、ジメチルアミノプロピル、ジメチルアミノブチルなどの炭素原子数1から6のアミノアルキル基；アミノ基等がC末端のカルボニル基に置換された誘導体などが挙げられる。本発明の蛍光試薬であるペプチドおよび式Ｉのペプチドの塩あるいはそれらののN末端もしくはC末端が置換された誘導体の塩としては、酢酸、トリフルオロ酢酸、クエン酸、リンゴ酸などの有機酸；塩酸、硫酸、リン酸などの無機酸等との酸付加塩が挙げられる。また、本発明の蛍光試薬であるペプチドまたは式Ｉのペプチドのカルボキシル基とのナトリウム塩、カリウム塩なども挙げられる。
【００１６】
本発明の式Ｉのペプチドの特に好ましい例としては、以下の式ＩＩで表されるペプチド、そのN末端もしくはC末端が置換されたその誘導体またはそれらの塩が挙げられる。
【化１５】

【００１７】
本発明の蛍光試薬は、反応性の官能基を有する適当なスペーサー鎖を介して蛍光色素基とインターカレート基を結合させることにより合成することができる。これらの合成反応は、それ自体周知の方法によって行うことができる。
本発明の蛍光試薬が、蛍光色素基で置換されたα−アミノ酸残基とインターカレート基で置換されたα−アミノ酸残基を含むα−アミノ酸残基からなるペプチドである場合には、そのようなペプチドの合成は、α−アミノ酸、蛍光色素基で置換されたα−アミノ酸およびインターカレート基で置換されたα−アミノ酸のそれぞれのアミノ基もしくはカルボキシ基の保護誘導体を、合成しようとするペプチドに対応させて、順次ペプチド結合により連結して行うこともでき、また周知の固相法もしくは液相法により合成することもでき、ペプチド合成機により合成することもできる。これらの合成法はそれ自体周知であり、周知の合成法を利用できる。あるいは、α−アミノ酸の保護誘導体と蛍光色素基で置換されたα−アミノ酸の保護誘導体またはインターカレート基で置換されたα−アミノ酸の保護誘導体とを反応させてペプチドを合成した後に、蛍光色素基またはインターカレート基を導入する反応を行って、本発明のペプチドを合成することもできる。これらの合成に用いるアミノ基もしくはカルボキシ基の保護基としては、上記したペプチドのN末端もしくはC末端が置換された誘導体について例示した官能基が挙げられる。
【００１８】
本発明の式Iのペプチドは、下記式IIIの蛍光色素基置換α−アミノ酸あるいはインターカレート基置換α-アミノ酸のアミノ基もしくはカルボニル基保護誘導体と下記式IVのα-アミノ酸のアミノ基もしくはカルボニル基保護誘導体とを用いて、周知のペプチド合成反応を行うことによって合成することができる。
【００１９】
【化１６】

ペプチド合成は、上記したと同様に、両者の保護誘導体を順次ペプチド結合により連結して行うこともでき、また周知の固相法もしくは液相法により合成することもでき、ペプチド合成機により合成することもできる。あるいは、式IVのα−アミノ酸の保護誘導体と式IIIの蛍光色素基で置換されたα−アミノ酸の保護誘導体またはインターカレート基で置換されたα−アミノ酸の保護誘導体とを反応させてペプチドを合成した後に、蛍光色素基またはインターカレート基を導入する反応を行って、式Iのペプチドを合成することもできる。
【００２０】
上記式IIIの蛍光色素基置換α-アミノ酸あるいはインターカレート基置換α-アミノ酸および上記式IVのα-アミノ酸のアミノ基もしくはカルボニル基保護誘導体としては、上記した保護基で保護されたものが挙げられる。上記式IIIの蛍光色素基置換α-アミノ酸あるいはインターカレート基置換α-アミノ酸は、下記式Vで表されるα-アミノ酸と下記式VIで表される蛍光色素誘導体あるいはインターカレーター誘導体(式VIにおいてDは、ハロゲン原子などの反応性基を表す)とを反応させることにより得ることができる。
【００２１】
【化１７】

式IVまたはVのα-アミノ酸としては、上記したアラニン、リシン、ノルリシン、アスパラギン酸、グルタミン、セリン、システインなどが挙げられる。
かくして式Iのペプチドを得ることができる。式IのペプチドのN末端もしくはC末端が置換されたその誘導体またはそれらの塩は、式Iのペプチドの合成の段階で得ることもでき、また式Iのペプチドからそれ自体周知の方法で得ることもできる。
式Iで表されるペプチド以外の本発明の蛍光試薬であるペプチドも、式Iのペプチドと同様に合成することができる。
【００２２】
本発明の蛍光試薬は、二本鎖DNA、二本鎖RNAなどの二本鎖核酸と接触することにより該蛍光試薬分子中のインターカレート基が二本鎖核酸の塩基対間にインターカレートして、該蛍光試薬分子中における蛍光色素基とインターカレート基とのスタッキングによる消光が解消して蛍光強度が増強し、それによって二本鎖核酸を高感度で選択的に検出することのできる。
本発明の蛍光試薬を用いて、二本鎖核酸を含むことが疑われる検体中の二本鎖核酸を検出するには、該蛍光試薬を検体と接触させて、該蛍光試薬の蛍光強度の増強度を測定することにより検出できる。蛍光試薬と検体を接触させる際の具体的条件、蛍光強度の具体的方法等は、それ自体周知の方法を採用することができる。本発明の蛍光試薬は、DNAチップまたはDNAマイクロアレイでのDNA検索に適用できる。即ち、サンプルDNAをDNAチップまたはDNAマイクロアレイに固定されたプローブNDAに反応させて、二本鎖DNA形成させ、この二本鎖DNAを本発明の蛍光試薬で高感度でかつ選択的に検出することができる。従って、本発明の蛍光試薬によれば、従来必要とされたサンプルDNAごとにラベル化試薬でラベル化する必要がなくなり、ラベル化剤の導入による二本鎖DNA形成能の低下が引き起こされたり、遺伝子の変異や多型の診断に誤った結果を導くこともない。
【００２３】
【実施例】
以下に本発明を実施例により更に詳細に説明するが、本発明はこれら実施例によって何ら限定されるものではない。
【００２４】
実施例１
FITC-K2-Acr2 の合成法
下記式で表される本発明の蛍光試薬である、インターカレート基としてアクリジン基、蛍光色素基としてフルオレセイン基が置換したリジンからなるペプチドを合成した。
【化１８】

【００２５】
１. Ac-Lys(Acr)-(Lys(Boc)) ₂ -Lys(Mtt)-(Lys(Boc)) ₂ -Lys(Acr)- 樹脂の合成
a) 方法
リアクションベッセルにFmoc-NH-SAL Resin 0.20g(0.5mmol/g)を入れ、カートリッジ1, 7にFmoc-Lys(Acr)-OHを0.21gずつ、カートリッジ2, 3, 5, 6にFmoc-Lys(Boc)-OHを0.19gずつ、カートリッジ4にFmoc-Lys(Mtt)-OHを0.25g入れた。後は、Applied Biosystems社の431Aペプチドシンセサイザーを用いて合成を行った。方法は、standard Fmoc法で行い、N末端はアセチル化した。
b) 結果
性状：黄色固体
収量：0.40g
【００２６】
２．Ac-Lys(Acr)-(Lys(Boc)) ₂ -Lys(Mtt)-(Lys(Boc)) ₂ -Lys(Acr)- 樹脂の Mtt 基の脱
保護、 FITC の修飾、レジンからの切り出しおよび側鎖の脱保護
a) 方法
Mtt基の脱保護：スクリュー管にレジン0.40gを入れ、これに過剰のジクロロメタン（DCM）を加えて30分かけて膨潤させた。その後、過剰のDCMを窒素ガスで除いた。その後、DCM:TFA:TIS（トリイソプロピルシラン）=94:1:5の混合溶液5mlを加えて2分攪拌し、窒素ガスで溶媒を除いた。この操作を3回繰り返して、吸引濾過しDCM、トリエチルアミン、DCMで洗浄後、減圧乾燥させた。
FITCの修飾：その後、NMP4mlを加えて30分間攪拌して膨潤させ、その後トリエチルアミン0.15mlを加えて攪拌した。それから、FITC 0.4gを加えて室温で12時間攪拌した（カイザーテスト）。その後吸引濾過し、NMP、DCMで洗浄して減圧乾燥させた。
樹脂からの切り出し及び側鎖の脱保護）：m-クレゾール 0.08ml、チオアニソール 0.48ml、TFA 3.44mlを加えて室温で1時間半撹拌した。その後、吸引濾過しTFAで洗浄した。TFAを減圧留去した後、氷浴中でエーテル15ml加えた。超音波照射後、しばらく放置し、上澄み液を取り除いた。次に、氷浴中で酢酸エチル15mlを加えて、超音波照射後、しばらく放置した。その後、吸引濾過しエーテルで洗浄後、減圧乾燥させた。
【００２７】
b) 結果
性状：黄橙色固体
収量：0.21g
【００２８】
３．Ac-Lys(Acr)-(Lys) ₂ -Lys(FITC)-(Lys) ₂ -Lys(Acr)-NH ₂ (FITC-K ₂ -Acr ₂ ) の HPLC による純度チェック
a) 方法
目的化合物の純度をチェックするために、HPLCによる分析を行った。条件は、以下の通りである。
検出波長：210nm
カラム：Inertsil ODS-3（4.6×250mm）
試料注入溶媒：超純水
流速：1ml/min
溶離液A:0.1%TFA水溶液
溶離液B:70%アセトニトリル/0.1%TFA溶液
グラジエント条件
【表１】

【００２９】
b) 結果
HPLCの精製により、R.T.=21minとR.T.=24min付近に2つの大きなピークが検出され、TOF MASSよりR.T.=21minのピークがFITCが修飾されてない化合物、R.T.=24minのピークが目的化合物であることが分かった。FITC修飾後のカイザーテスト（樹脂上のアミノ基をニンヒドリンにより検出する方法）で99%以上FITCが反応したという結果が得られた。
精製されたFITC-K₂-Acr₂のHPLCチャートを図１に示した。また、精製されたFITC-K₂-Acr₂のTOF MASSデータを図２に示した。
【００３０】
実施例２
蛍光スペクトル測定
二本鎖DNA、一本鎖DNAを添加していった時のFITC-K₂-Acr₂の蛍光スペクトルを測定し、二本鎖DNAに対して有効に機能するかどうかを調べた。
１. Calf thymus DNA 添加に伴う FITC-K ₂ -Acr ₂ の蛍光スペクトル測定
a) 方法
以下の条件で、本発明の蛍光試薬であるFITC-K₂-Acr₂に二本鎖DNAであるCalf thymus DNA添加して蛍光スペクトルを測定した。
測定溶液：10mM Tris-HCl, 1mM EDTA(pH8.3), 0.2M NaCl, 25℃
セル中の色素濃度：0.16μM、[ct DNA]=0.25mM(-bp)、[dye]=0.1mM
EX Slit:10nm, EM Slit:5nm、λex=492nm
b) 結果
得られた蛍光スペクトルを図３に示した。図３から分かるように、Tris-HCl buffer(pH8.3)の場合は、約25倍の蛍光強度の増大が起きた。
【００３１】
２. poly[d(A-T)] ₂ 添加に伴う FITC-K ₂ -Acr ₂ の蛍光スペクトル測定
a) 方法
上記１と同様にTris-HCl buffer(pH8.3)を用い以下の条件で、poly[d(A-T)]₂添加に伴うFITC-K₂-Acr₂の蛍光スペクトルを測定した。
測定溶液：10mM Tris-HCl, 1mM EDTA(pH8.3), 0.2M NaCl, 25℃
セル中の色素濃度：0.16μM、[poly[d(A-T)]₂]=1.10mM(-bp)、
[dye]=0.1mM
EX Slit:10nm, EM Slit:5nm
b) 結果
得られた蛍光スペクトルを図４に示した。ほとんど図３のct DNAの場合と同様の結果が得られた。
【００３２】
３. poly[d(G-C)] ₂ 添加に伴う FITC-K ₂ -Acr ₂ の蛍光スペクトル測定a) 方法
上記１と同様に、以下の条件で、poly[d(G-C)]₂添加に伴うFITC-K₂-Acr₂の蛍光スペクトルを測定した。
測定溶液：10mM Tris-HCl, 1mM EDTA(pH8.3), 0.2M NaCl, 25℃
セル中の色素濃度：0.16μM、[poly[d(G-C)]₂]=1.01mM(-bp)、
[dye]=0.1mM
EX Slit:10nm, EM Slit:5nm
b) 結果
得られた蛍光スペクトルを図５に示した。ct DNA、poly[d(A-T)]₂と同様の結果が得られた。
【００３３】
４．polydT 添加に伴う FITC-K ₂ -Acr ₂ の蛍光スペクトル測定
a) 方法
以下の条件で、一本鎖DNAであるpolydTを添加してFITC-K₂-Acr₂の蛍光スペクトルを測定した。
測定溶液：10mM Tris-HCl, 1mM EDTA(pH8.3), 0.2M NaCl, 25℃
セル中の色素濃度：0.16μM、[polydT]=1.07mM(-b)、[dye]=0.1mM
EX Slit:10nm, EM Slit:5nm
b) 結果
得られた蛍光スペクトルを図６に示した。一本鎖 DNAであるpolydT添加に伴ってほとんで蛍光強度は変化しなかった。
【００３４】
５. polydA 添加に伴う FITC-K ₂ -Acr ₂ の蛍光スペクトル測定
同様に以下の条件で、一本鎖DNAであるpolydAを添加してFITC-K₂-Acr₂の蛍光スペクトルを測定した。
測定溶液：10mM Tris-HCl, 1mM EDTA(pH8.3), 0.2M NaCl, 25℃
セル中の色素濃度：0.16μM、[polydA]=1.04mM(-b)、[dye]=0.1mM
EX Slit:10nm, EM Slit:5nm
b) 結果
得られた蛍光スペクトルを図７に示した。一本鎖DNAであるpolydA添加に伴ってもほとんで蛍光強度は変化しなかった。
【００３５】
従って、FITC-K₂-Acr₂は二本鎖DNAと結合することによって、その蛍光強度は20〜25倍増大するが、一本鎖DNAを添加してもほとんど蛍光強度は変化しなかった。
【００３６】
【発明の効果】
以上の結果より、二本鎖核酸を選択的に検出できる本発明の蛍光色素としてFITC-K₂-Acr₂が有用であるということが示された。本発明の蛍光試薬の蛍光メカニズムは従来にない概念に基くものである。本発明の蛍光試薬の蛍光メカニズムを図８の模式図に示した。即ち、本発明の蛍光色素基およびインターカレート基を含有する蛍光試薬は、二本鎖核酸と接触することにより該蛍光試薬分子中のインターカレート基が二本鎖核酸の塩基対間にインターカレートして、該蛍光試薬分子中における蛍光色素基とインターカレート基とのスタッキングによる消光が解消して蛍光強度が増強し、それによって二本鎖核酸を選択的に検出することのできる。
【図面の簡単な説明】
【図１】図１は、本発明の蛍光試薬である精製されたFITC-K₂-Acr₂HPLCチャート示す。
【図２】図２は、本発明の蛍光試薬である精製されたFITC-K₂-Acr₂のTOF MASSデータを示す。
【図３】図３は、本発明の蛍光試薬であるFITC-K₂-Acr₂に二本鎖DNAであるCalf thymus DNAを添加して測定した蛍光スペクトルを示す。
【図４】図４は、本発明の蛍光試薬であるFITC-K₂-Acr₂に二本鎖DNAであるpoly[d(A-T)]₂を添加して測定した蛍光スペクトルを示す。
【図５】図５は、本発明の蛍光試薬であるFITC-K₂-Acr₂に二本鎖DNAであるpoly[d(G-C)]₂を添加して測定した蛍光スペクトルを示す。
【図６】図６は、本発明の蛍光試薬であるFITC-K₂-Acr₂に一本鎖DNAであるpolydTを添加して測定した蛍光スペクトルを示す。
【図７】図７は、本発明の蛍光試薬であるFITC-K₂-Acr₂に一本鎖DNAであるpolydA を添加して測定した蛍光スペクトルを示す。
【図８】図８は、本発明の蛍光試薬の蛍光メカニズムを示す模式図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a fluorescent reagent for selectively detecting a double-stranded nucleic acid containing a fluorescent dye group and an intercalate group. More specifically, a fluorescent reagent capable of selectively detecting a double-stranded nucleic acid containing a fluorescent dye group and an intercalating group, and intercalating in the fluorescent reagent molecule by contacting with the double-stranded nucleic acid The group intercalates between the base pairs of the double-stranded nucleic acid, quenching by the stacking of the fluorescent dye group and the intercalating group in the fluorescent reagent molecule is eliminated, and the fluorescence intensity is thereby increased. The present invention relates to a fluorescent reagent for selectively detecting a double-stranded nucleic acid containing a fluorescent dye group and an intercalating group, which can selectively detect a strand nucleic acid. According to the fluorescent reagent of the present invention, for example, when a target DNA is detected by a DNA chip or a DNA microarray, the target DNA is reacted with the DNA chip or the DNA microarray as it is without labeling, thereby forming a double strand. DNA can be detected directly and selectively.
[0002]
[Prior art]
It is known that DNA, which is genetic material of biological cells, interacts with certain drugs, carcinogens, mutagens, fluorescent dyes and the like. Because DNA plays a central role in replication and protein synthesis in organisms, such interaction modifications can significantly alter cellular metabolism, weaken cell growth, or even stop it in some cases. Sometimes.
In the 21st century, the entire human genome with a length of 3 billion base pairs has been revealed. Along with this, the development of a rapid and sensitive detection method for specific genes will be developed from the viewpoint of gene therapy and genetic diagnosis. It is becoming increasingly important. Research on DNA chips or DNA microarrays in which hundreds to hundreds of thousands of probe DNAs are arranged in a spot or array on a solid surface of about 1 cm 2 has been conducted worldwide as a post-genome.
Conventional detection methods using a DNA chip or DNA microarray introduce a labeling agent such as a fluorescent reagent into the target DNA and detect the double-stranded DNA formed by reacting this with the DNA chip or DNA microarray using the labeling agent. To do.
[0003]
[Problems to be solved by the invention]
However, in the case of such a conventional detection method, it is necessary to carry out a labeling reaction for each sample DNA, and the introduction of a labeling agent causes a decrease in the ability to form double-stranded DNA. It will lead to false results in the diagnosis of mutations and polymorphisms.
Therefore, the object of the present invention is to eliminate the need to introduce a labeling agent for each sample DNA when detecting sample DNA using a DNA chip or DNA microarray. The purpose of the present invention is to provide a completely new fluorescent reagent that allows a double-stranded DNA to be selectively detected by adding an analytical reagent directly after hybridization.
Another object of the present invention is to provide a method for selectively detecting a double-stranded nucleic acid using this new fluorescent reagent.
Still another object of the present invention is to provide a new peptide used as this new fluorescent reagent.
[0004]
[Means for Solving the Problems]
As a result of intensive research aimed at obtaining a fluorescent reagent for achieving the above-mentioned object, the present inventor has found that a fluorescent reagent containing a fluorescent dye group and an intercalating group is in the state where the fluorescent reagent is used alone. Fluorescence is quenched by stacking the fluorescent dye group and intercalating group in the fluorescent reagent molecule, but the intercalating group intercalates between the base pairs of the double-stranded DNA by contacting with the double-stranded DNA. In this way, we have found a completely new concept of a fluorescent reagent that eliminates quenching due to stacking of fluorescent dye groups and intercalating groups and enhances fluorescence intensity, thereby selectively detecting double-stranded DNA. Based on this knowledge, further research was carried out to complete the present invention.
[0005]
Accordingly, the present invention provides a fluorescent reagent capable of selectively detecting a double-stranded nucleic acid containing a fluorescent dye group and an intercalating group, and the intercalation in the fluorescent reagent molecule by contacting with the double-stranded nucleic acid. Group is intercalated between the base pairs of the double-stranded nucleic acid, quenching due to stacking of the fluorescent dye group and the intercalating group in the fluorescent reagent molecule is eliminated, and the fluorescence intensity is increased. The present invention relates to a fluorescent reagent for selectively detecting a double-stranded nucleic acid containing a fluorescent dye group and an intercalating group, which can selectively detect a double-stranded nucleic acid.
Furthermore, the present invention is a method for selectively detecting double-stranded nucleic acid in a specimen, wherein the fluorescent reagent is brought into contact with a specimen suspected of containing double-stranded nucleic acid, and the fluorescence intensity of the fluorescent reagent is increased. The present invention relates to a method for selectively detecting double-stranded nucleic acid in a sample by enhancement.
Furthermore, the present invention relates to a peptide represented by the following formula I that can be used as the fluorescent reagent, a derivative thereof substituted at the N-terminus or C-terminus, or a salt thereof.
[Formula 4]

(Wherein X represents an intercalating group or fluorescent dye group, Y represents a divalent group constituting an α-amino acid, Z represents a monovalent group constituting an α-amino acid, and n represents 1 Represents an integer of 8 to 8. However, each X in the formula I may be different from or the same fluorescent dye group or intercalating group, and in the formula I, always an intercalating group and a fluorescent dye group. Both are present, and each Y and Z in formula I may be different or the same).
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The fluorescent reagent of the present invention is a fluorescent reagent that can selectively detect a double-stranded nucleic acid containing a fluorescent dye group and an intercalating group, and is brought into contact with the double-stranded nucleic acid to thereby intercalate in the fluorescent reagent molecule. The calate group intercalates between the base pairs of the double-stranded nucleic acid, quenching due to stacking of the fluorescent dye group and the intercalate group in the fluorescent reagent molecule is eliminated, and the fluorescence intensity is thereby enhanced. It is a fluorescent reagent that can selectively detect double-stranded nucleic acids.
The fluorescent reagent of the present invention has a fluorescent dye group and an intercalating group in its molecule, causing a quenching phenomenon by stacking them, and intercalating by contacting with a double-stranded nucleic acid. Any group may be used as long as the group intercalates between the base pairs of the double-stranded nucleic acid so that quenching can be eliminated. Examples of such a fluorescent reagent of the present invention include a fluorescent reagent in which a fluorescent dye group and an intercalating group are bonded via an appropriate spacer chain having a reactive functional group.
[0007]
The fluorescent reagent of the present invention is preferably a peptide comprising an α-amino acid residue containing an α-amino acid residue substituted with a fluorescent dye group and an α-amino acid residue substituted with an intercalating group. That is, a peptide in which a fluorescent dye group and an intercalating group are substituted for a peptide consisting of an α-amino acid residue is preferable. Furthermore, it is preferable that the fluorescent dye group and the intercalate group are substituted every 2 to 10, more preferably 2 to 5, especially 2 α-amino acid residues. That is, the α-amino acid residue substituted with the fluorescent dye group and the α-amino acid residue substituted with the intercalating group have 2 to 10, more preferably 2 to 5, particularly 2 α-amino acid residues. It is preferably present every other group. The α-amino acid residue substituted with a fluorescent dye group is preferably present between α-amino acid residues substituted with an intercalating group. In such a case, the intercalating group effectively intercalates between the base pairs of the double-stranded nucleic acid, and quenching due to stacking of the fluorescent dye group and the intercalating group in the fluorescent reagent molecule is prevented. It effectively eliminates and the fluorescence intensity is remarkably enhanced, so that double-stranded nucleic acid can be selectively detected. In addition, the peptide which is the fluorescent reagent of the present invention is preferably a peptide having a total α-amino acid residue number of 4 to 40, particularly 4 to 25. Furthermore, peptides having 4 to 16 total α-amino acid residues, particularly 4 to 10 are preferred.
In the present invention, the fluorescent dye group is preferably a fluorescent dye group that does not have a function as an intercalator.
[0008]
More specifically, the fluorescent reagent of the present invention is preferably a peptide represented by the following formula I, a derivative thereof substituted at its N-terminal or C-terminal, or a salt thereof.
[Chemical formula 5]

(Wherein X represents an intercalating group or fluorescent dye group, Y represents a divalent group constituting an α-amino acid, Z represents a monovalent group constituting an α-amino acid, and n represents 1 Represents an integer of 8 to 8. However, each X in the formula I may be different from or the same fluorescent dye group or intercalating group, and in the formula I, always an intercalating group and a fluorescent dye group. Both are present, and each Y and Z in formula I may be different or the same).
The peptide represented by the above formula I and its derivative substituted at its N-terminal or C-terminal are novel compounds.
[0009]
As the intercalating group in the fluorescent reagent of the present invention and the intercalating group of X in formula I, any one can be used as long as it can be inserted in parallel between the base pairs of double-stranded DNA or double-stranded RNA. It may be a group of Examples of such an intercalating group include a group having an acridine skeleton which may have a substituent, a group having an anthraquinone skeleton which may have a substituent, and an anthracene which may have a substituent. A group having a skeleton, a group having a naphthaleneimide skeleton optionally having a substituent, a group having a naphthalenediimide skeleton optionally having a substituent, and a pyrene skeleton optionally having a substituent Groups and the like. Examples of the substituent herein include halogen atoms such as chlorine, fluorine and bromine; and carbon atoms which may be substituted with halogen atoms such as methyl, ethyl, propyl, butyl, pentyl, hexyl and trifluoromethyl. Alkyl groups having 1 to 6 carbon atoms; alkoxy groups having 1 to 6 carbon atoms such as methoxy, ethoxy, butoxy, pentoxy, hexoxy; acyl groups having 1 to 7 carbon atoms such as formyl, acetyl, butanoyl, pentanoyl, hexanoyl; acetyloxy Acyloxy groups having 2 to 7 carbon atoms such as butanoyloxy, pentanoyloxy, hexanoyloxy; aminomethyl, aminoethyl, aminopropyl, methylaminomethyl, dimethylamimethyl, dimethylaminopropyl, dimethylaminobutyl, etc. Alkyl group having 1 to 6 carbon atoms Is an aminoalkyl group having 1 to 6 carbon atoms which may be substituted by 1 or 2. These substituents may be substituted at any position of an acridine skeleton, anthraquinone skeleton, anthracene skeleton, or the like, and two or more plural substituents may be substituted with these skeletons.
In addition, the acridine skeleton, anthraquinone skeleton, anthracene skeleton and the like may have a functional group for binding to Y in the fluorescent reagent of the present invention and the peptide of formula I. As such a functional group, For example, -CO-, -NH-, -SO₂-, -PO₂-, -S-, -N (NH) NH-, and groups in which these functional groups are bonded to the terminal or side chain of an alkylene group having 1 to 6 carbon atoms.
[0010]
A group having an acridine skeleton optionally having a substituent, a group having an anthraquinone skeleton optionally having a substituent, a group having an anthracene skeleton optionally having a substituent, and a substituent; Specific examples of the group having a naphthalene imide skeleton which may have, a group having a naphthalenediimide skeleton which may have a substituent, and a group having a pyrene skeleton which may have a substituent are as follows. Can be mentioned.
[0011]
A group having an acridine skeleton which may have a substituent
[Chemical 6]

A group having an anthraquinone skeleton which may have a substituent
[Chemical 7]

A group having an anthracene skeleton which may have a substituent
[Chemical 8]

A group having a naphthaleneimide skeleton which may have a substituent
[Chemical 9]

A group having a naphthalenediimide skeleton which may have a substituent
[Chemical Formula 10]

A group having a pyrene skeleton which may have a substituent
Embedded image

[0012]
The fluorescent dye group in the fluorescent reagent of the present invention and the X fluorescent dye group in Formula I are not particularly limited as long as they are fluorescent groups, and any group may be used. In the present invention, the fluorescent dye group is preferably a fluorescent dye group that does not have a function as an intercalator. Examples of such a fluorescent dye group include a group having a xanthene skeleton which may have a substituent, a group having an indole skeleton which may have a substituent, and a substituent. Examples thereof include a group having a boron-containing and nitrogen indacene skeleton. Examples of the substituent herein include the same substituents as those in the case of the above-described intercalate group. Furthermore, a carboxy group, a sulfonic acid group, an imino group, or a group in which these functional groups are bonded to the terminal or side chain of an alkylene group having 1 to 6 carbon atoms. In the case of a group having a xanthene skeleton, a substituent having a form in which a quinolizidine ring, a pyrrole ring, or the like is condensed is exemplified. In the case of a group having an indole skeleton, two indole skeletons may be bonded via an alkylene group having 1 to 6 carbon atoms, an alkenylene group, or the like. In the case of a group having a boron-containing and nitrogen indacene skeleton, a phenyl group or a phenyl group substituted with the same substituent as the substituent in the case of the intercalate group described above may be substituted.
Further, similarly to the case of the intercalating group described above, it may have a functional group for binding to Y in the fluorescent reagent of the present invention and the peptide of formula I.
Specific examples of such fluorescent dye groups include the following.
[0013]
Group having xanthene skeleton which may have a substituent
Embedded image

A group having an indole skeleton which may have a substituent
Embedded image

A group having a boron-containing and nitrogen-indacene skeleton, which may have a substituent
Embedded image

[0014]
In Formula I, each X may be a different or the same fluorescent dye group or intercalating group, and in Formula I, both an intercalating group and a fluorescent dye group are always present. In particular, in the peptide of formula I, the fluorescent dye group and the intercalating group are preferably present adjacent to each other, and in particular, a peptide having a structure in which an intercalating group exists on both sides of the fluorescent dye group. preferable. In Formula I, Y represents a divalent group (having two bonds) constituting an α-amino acid, and Z represents a monovalent group (having one bond) constituting an α-amino acid. That is, Y is changed from α-amino acid to -CH (NH₂) Represents a divalent group constituting the remaining α-amino acid excluding COOH, and Z represents a monovalent group. Specifically, Y is a group represented by -AW-, wherein A represents a linear or branched alkylene group having 1 to 6 carbon atoms such as methylene, ethylene, propylene, methylmethylene, W represents a single bond, NH, CO, CONH, NHCO, O, S, or HNC (NH) NH. Z is a group represented by A—W—H. These Y and Z are α-amino acids such as alanine, lysine, norlysine, aspartic acid, glutamine, serine, cysteine, etc.₂) Each of the α-amino acids other than COOH represents a divalent group or a monovalent group. Each Y and Z in Formula I may be different from each other or the same. In the peptide of formula I, n represents an integer from 1 to 8. Preferably n is an integer from 1 to 5, particularly preferably n is an integer from 1 to 3.
[0015]
The peptide which is a fluorescent reagent of the present invention and the peptide of formula I may be a derivative thereof or a salt thereof substituted at its N-terminus or C-terminus. Examples of derivatives substituted at the N-terminus include acyl groups having 1 to 7 carbon atoms such as formyl, acetyl, butanoyl, pentanoyl, hexanoyl; methoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, pentyloxycarbonyl, hexyloxy Examples thereof include derivatives in which an alkoxycarbonyl group having 2 to 7 carbon atoms such as carbonyl is substituted with an N-terminal amino group. Derivatives substituted at the C-terminus include alkyl groups having 1 to 6 carbon atoms which may be substituted with halogen atoms such as methyl, ethyl, propyl, butyl, pentyl, hexyl, trifluoromethyl; aminomethyl, amino Aminoalkyl groups having 1 to 6 carbon atoms, such as ethyl, aminopropyl, methylaminomethyl, dimethylaminomethyl, dimethylaminopropyl, dimethylaminobutyl; derivatives in which the amino group or the like is substituted with a C-terminal carbonyl group It is done. Examples of the salt of the peptide which is the fluorescent reagent of the present invention and the peptide of the formula I or a derivative thereof substituted at the N-terminal or C-terminal thereof include organic acids such as acetic acid, trifluoroacetic acid, citric acid and malic acid; Acid addition salts with inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid. Moreover, the sodium salt with the carboxyl group of the peptide which is the fluorescent reagent of this invention, or the peptide of Formula I, potassium salt, etc. are mentioned.
[0016]
Particularly preferred examples of the peptide of formula I of the present invention include the peptide represented by the following formula II, a derivative thereof substituted at the N-terminus or C-terminus, or a salt thereof.
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[0017]
The fluorescent reagent of the present invention can be synthesized by combining a fluorescent dye group and an intercalating group via an appropriate spacer chain having a reactive functional group. These synthesis reactions can be carried out by a method known per se.
When the fluorescent reagent of the present invention is a peptide comprising an α-amino acid residue containing an α-amino acid residue substituted with a fluorescent dye group and an α-amino acid residue substituted with an intercalating group, The synthesis of such peptides seeks to synthesize protected derivatives of the respective amino or carboxy groups of α-amino acids, α-amino acids substituted with fluorescent dye groups and α-amino acids substituted with intercalating groups Corresponding to peptides, they can be sequentially linked by peptide bonds, synthesized by a well-known solid phase method or liquid phase method, and synthesized by a peptide synthesizer. These synthesis methods are known per se, and known synthesis methods can be used. Alternatively, after a peptide is synthesized by reacting a protected derivative of an α-amino acid with a protected derivative of an α-amino acid substituted with a fluorescent dye group or a protected derivative of an α-amino acid substituted with an intercalating group, the fluorescent dye A peptide of the present invention can also be synthesized by carrying out a reaction for introducing a group or an intercalate group. Examples of the protecting group for the amino group or carboxy group used in these syntheses include the functional groups exemplified for the above-described derivatives in which the N-terminal or C-terminal of the peptide is substituted.
[0018]
The peptide of the formula I of the present invention comprises an amino group or carbonyl group-protected derivative of a fluorescent dye group-substituted α-amino acid or intercalate group-substituted α-amino acid of the following formula III and an amino group or carbonyl of the α-amino acid of the following formula IV: It can synthesize | combine by performing a well-known peptide synthesis reaction using a group protection derivative.
[0019]
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In the same manner as described above, peptide synthesis can be performed by sequentially linking both protected derivatives by peptide bonds, or by a well-known solid phase method or liquid phase method, and by a peptide synthesizer. You can also Alternatively, the peptide is obtained by reacting a protected derivative of an α-amino acid of formula IV with a protected derivative of an α-amino acid substituted with a fluorescent dye group of formula III or a protected derivative of an α-amino acid substituted with an intercalating group. After the synthesis, a peptide of formula I can also be synthesized by carrying out a reaction for introducing a fluorescent dye group or an intercalate group.
[0020]
Examples of the fluorescent dye group-substituted α-amino acid or intercalate group-substituted α-amino acid of the above formula III and the amino group or carbonyl group protected derivative of the α-amino acid of the above formula IV include those protected with the above-described protecting groups. It is done. The fluorescent dye group-substituted α-amino acid or intercalate group-substituted α-amino acid of the above formula III includes an α-amino acid represented by the following formula V and a fluorescent dye derivative or intercalator derivative represented by the following formula VI (formula VI D can be obtained by reacting with a reactive group such as a halogen atom.
[0021]
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Examples of the α-amino acid of formula IV or V include alanine, lysine, norlysine, aspartic acid, glutamine, serine, cysteine and the like described above.
A peptide of formula I can thus be obtained. A derivative thereof or a salt thereof in which the N-terminus or C-terminus of the peptide of formula I is substituted can also be obtained at the stage of synthesis of the peptide of formula I, or from a peptide of formula I in a manner known per se. You can also.
Peptides that are fluorescent reagents of the present invention other than the peptide represented by Formula I can be synthesized in the same manner as the peptide of Formula I.
[0022]
When the fluorescent reagent of the present invention is brought into contact with a double-stranded nucleic acid such as double-stranded DNA or double-stranded RNA, the intercalating group in the fluorescent reagent molecule is intercalated between the base pairs of the double-stranded nucleic acid. Thus, quenching due to stacking of the fluorescent dye group and the intercalating group in the fluorescent reagent molecule is eliminated, and the fluorescence intensity is enhanced, whereby a double-stranded nucleic acid can be selectively detected with high sensitivity. .
In order to detect double-stranded nucleic acid in a specimen suspected of containing double-stranded nucleic acid using the fluorescent reagent of the present invention, the fluorescent reagent is brought into contact with the specimen to increase the fluorescence intensity of the fluorescent reagent. It can be detected by measuring the degree. As specific conditions for bringing the fluorescent reagent into contact with the specimen, specific methods of fluorescence intensity, etc., methods known per se can be employed. The fluorescent reagent of the present invention can be applied to DNA search using a DNA chip or a DNA microarray. That is, sample DNA is reacted with a probe NDA immobilized on a DNA chip or DNA microarray to form double-stranded DNA, and this double-stranded DNA is selectively detected with high sensitivity by the fluorescent reagent of the present invention. Can do. Therefore, according to the fluorescent reagent of the present invention, there is no need to label with a labeling reagent for each sample DNA conventionally required, causing a decrease in the ability to form double-stranded DNA by introducing a labeling agent, It does not lead to false results in the diagnosis of genetic mutations and polymorphisms.
[0023]
【Example】
Examples The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
[0024]
Example 1
FITC-K2-Acr2 Synthesis method of
A peptide consisting of lysine substituted with an acridine group as an intercalating group and a fluorescein group as a fluorescent dye group, which is a fluorescent reagent of the present invention represented by the following formula, was synthesized.
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[0025]
1.Ac-Lys (Acr)-(Lys (Boc)) ₂ -Lys (Mtt)-(Lys (Boc)) ₂ -Lys (Acr)- Resin synthesis
a) Method
Add 0.20 g (0.5 mmol / g) of Fmoc-NH-SAL Resin to the reaction vessel, 0.21 g of Fmoc-Lys (Acr) -OH in cartridges 1 and 7, and Fmoc-Lys in cartridges 2, 3, 5, 6 0.19 g of (Boc) -OH was added to each cartridge, and 0.25 g of Fmoc-Lys (Mtt) -OH was added to cartridge 4. Thereafter, synthesis was performed using a 431A peptide synthesizer manufactured by Applied Biosystems. The method was performed by the standard Fmoc method, and the N-terminal was acetylated.
b) Results
Property: Yellow solid
Yield: 0.40g
[0026]
2.Ac-Lys (Acr)-(Lys (Boc)) ₂ -Lys (Mtt)-(Lys (Boc)) ₂ -Lys (Acr)- Resin Mtt Group removal
protection, FITC Modification, resin excision and side chain deprotection
a) Method
Deprotection of Mtt group: 0.40 g of resin was placed in a screw tube, and excess dichloromethane (DCM) was added thereto to swell for 30 minutes. Thereafter, excess DCM was removed with nitrogen gas. Thereafter, 5 ml of a mixed solution of DCM: TFA: TIS (triisopropylsilane) = 94: 1: 5 was added and stirred for 2 minutes, and the solvent was removed with nitrogen gas. This operation was repeated three times, suction filtered, washed with DCM, triethylamine and DCM, and then dried under reduced pressure.
Modification of FITC: After that, 4 ml of NMP was added and stirred for 30 minutes to swell, and then 0.15 ml of triethylamine was added and stirred. Then, 0.4 g of FITC was added and stirred at room temperature for 12 hours (Kaiser test). Thereafter, the solution was filtered with suction, washed with NMP and DCM, and dried under reduced pressure.
Cleavage from resin and deprotection of side chain): 0.08 ml of m-cresol, 0.48 ml of thioanisole and 3.44 ml of TFA were added, and the mixture was stirred at room temperature for 1.5 hours. Thereafter, the solution was suction filtered and washed with TFA. TFA was distilled off under reduced pressure, and 15 ml of ether was added in an ice bath. After ultrasonic irradiation, the solution was left for a while and the supernatant was removed. Next, 15 ml of ethyl acetate was added in an ice bath and left for a while after ultrasonic irradiation. Thereafter, the solution was suction filtered, washed with ether, and dried under reduced pressure.
[0027]
b) Results
Properties: Yellow-orange solid
Yield: 0.21g
[0028]
3.Ac-Lys (Acr)-(Lys) ₂ -Lys (FITC)-(Lys) ₂ -Lys (Acr) -NH ₂ (FITC-K ₂ -Acr ₂ ) of HPLC Purity check by
a) Method
In order to check the purity of the target compound, analysis by HPLC was performed. The conditions are as follows.
Detection wavelength: 210nm
Column: Inertsil ODS-3 (4.6 x 250 mm)
Sample injection solvent: ultrapure water
Flow rate: 1ml / min
Eluent A: 0.1% TFA aqueous solution
Eluent B: 70% acetonitrile / 0.1% TFA solution
Gradient condition
[Table 1]

[0029]
b) Results
After purification by HPLC, two large peaks are detected around RT = 21min and RT = 24min. From TOF MASS, RT = 21min peak is the compound without FITC modification, and RT = 24min peak is the target compound. I understood. In the Kaiser test after FITC modification (method of detecting amino groups on the resin with ninhydrin), it was found that more than 99% of FITC reacted.
Purified FITC-K₂-Acr₂The HPLC chart is shown in FIG. In addition, purified FITC-K₂-Acr₂Fig. 2 shows the TOF MASS data.
[0030]
Example 2
Fluorescence spectrum measurement
FITC-K when double-stranded DNA or single-stranded DNA is added₂-Acr₂The fluorescence spectrum was measured to determine whether it functions effectively on double-stranded DNA.
1.Calf thymus DNA Accompanying addition FITC-K ₂ -Acr ₂ Fluorescence spectrum measurement
a) Method
FITC-K which is the fluorescent reagent of the present invention under the following conditions₂-Acr₂The fluorescence spectrum was measured by adding Calf thymus DNA, which is a double-stranded DNA.
Measurement solution: 10 mM Tris-HCl, 1 mM EDTA (pH 8.3), 0.2 M NaCl, 25 ° C.
Dye concentration in the cell: 0.16 μM, [ct DNA] = 0.25 mM (−bp), [dye] = 0.1 mM
EX Slit: 10nm, EM Slit: 5nm, λex = 492nm
b) Results
The obtained fluorescence spectrum is shown in FIG. As can be seen from FIG. 3, in the case of Tris-HCl buffer (pH 8.3), the fluorescence intensity increased about 25 times.
[0031]
2.poly [d (AT)] ₂ Accompanying addition FITC-K ₂ -Acr ₂ Fluorescence spectrum measurement
a) Method
Poly [d (A-T)] using Tris-HCl buffer (pH 8.3) as in 1 above under the following conditions₂FITC-K with addition₂-Acr₂The fluorescence spectrum of was measured.
Measurement solution: 10 mM Tris-HCl, 1 mM EDTA (pH 8.3), 0.2 M NaCl, 25 ° C.
Dye concentration in the cell: 0.16 μM, [poly [d (A-T)]₂] = 1.10mM (-bp),
[dye] = 0.1mM
EX Slit: 10nm, EM Slit: 5nm
b) Results
The obtained fluorescence spectrum is shown in FIG. Almost the same result as in the case of ct DNA in FIG. 3 was obtained.
[0032]
3.poly [d (GC)] ₂ Accompanying addition FITC-K ₂ -Acr ₂ Fluorescence spectrum measurementa) Method
As in 1 above, poly [d (G-C)] under the following conditions:₂FITC-K with addition₂-Acr₂The fluorescence spectrum of was measured.
Measurement solution: 10 mM Tris-HCl, 1 mM EDTA (pH 8.3), 0.2 M NaCl, 25 ° C.
Dye concentration in the cell: 0.16 μM, [poly [d (G-C)]₂] = 1.01mM (-bp),
[dye] = 0.1mM
EX Slit: 10nm, EM Slit: 5nm
b) Results
The obtained fluorescence spectrum is shown in FIG. ct DNA, poly [d (A-T)]₂Similar results were obtained.
[0033]
4).polydT Accompanying addition FITC-K ₂ -Acr ₂ Fluorescence spectrum measurement
a) Method
Under the following conditions, add single-stranded DNA polydT and add FITC-K₂-Acr₂The fluorescence spectrum of was measured.
Measurement solution: 10 mM Tris-HCl, 1 mM EDTA (pH 8.3), 0.2 M NaCl, 25 ° C.
Dye concentration in the cell: 0.16 μM, [polydT] = 1.07 mM (-b), [dye] = 0.1 mM
EX Slit: 10nm, EM Slit: 5nm
b) Results
The obtained fluorescence spectrum is shown in FIG. The fluorescence intensity did not change with the addition of polydT, a single-stranded DNA.
[0034]
5.polydA Accompanying addition FITC-K ₂ -Acr ₂ Fluorescence spectrum measurement
Similarly, under the conditions below, add single-stranded DNA polydA and add FITC-K.₂-Acr₂The fluorescence spectrum of was measured.
Measurement solution: 10 mM Tris-HCl, 1 mM EDTA (pH 8.3), 0.2 M NaCl, 25 ° C.
Dye concentration in the cell: 0.16 μM, [polydA] = 1.04 mM (-b), [dye] = 0.1 mM
EX Slit: 10nm, EM Slit: 5nm
b) Results
The obtained fluorescence spectrum is shown in FIG. Even with the addition of polydA, a single-stranded DNA, the fluorescence intensity remained almost unchanged.
[0035]
Therefore, FITC-K₂-Acr₂By binding to double-stranded DNA, the fluorescence intensity increased 20 to 25 times, but even when single-stranded DNA was added, the fluorescence intensity hardly changed.
[0036]
【The invention's effect】
From the above results, FITC-K as the fluorescent dye of the present invention capable of selectively detecting double-stranded nucleic acids.₂-Acr₂Has been shown to be useful. The fluorescence mechanism of the fluorescent reagent of the present invention is based on an unconventional concept. The fluorescence mechanism of the fluorescent reagent of the present invention is shown in the schematic diagram of FIG. That is, the fluorescent reagent containing the fluorescent dye group and the intercalating group of the present invention is brought into contact with the double-stranded nucleic acid so that the intercalating group in the fluorescent reagent molecule is intercalated between the base pairs of the double-stranded nucleic acid. By quenching, quenching due to stacking of the fluorescent dye group and the intercalating group in the fluorescent reagent molecule is eliminated and the fluorescence intensity is enhanced, whereby the double-stranded nucleic acid can be selectively detected.
[Brief description of the drawings]
FIG. 1 shows purified FITC-K which is a fluorescent reagent of the present invention.₂-Acr₂An HPLC chart is shown.
FIG. 2 shows purified FITC-K, which is a fluorescent reagent of the present invention.₂-Acr₂Shows TOF MASS data.
FIG. 3 shows FITC-K which is the fluorescent reagent of the present invention.₂-Acr₂Shows a fluorescence spectrum measured by adding Calf thymus DNA, which is a double-stranded DNA.
FIG. 4 shows FITC-K which is a fluorescent reagent of the present invention.₂-Acr₂Poly [d (A-T)], a double-stranded DNA₂Shows a fluorescence spectrum measured by adding.
FIG. 5 shows FITC-K, which is a fluorescent reagent of the present invention.₂-Acr₂Poly [d (G-C)], a double-stranded DNA₂Shows a fluorescence spectrum measured by adding.
FIG. 6 shows FITC-K which is the fluorescent reagent of the present invention.₂-Acr₂Shows the fluorescence spectrum measured by adding polydT, which is a single-stranded DNA.
FIG. 7 shows FITC-K, which is a fluorescent reagent of the present invention.₂-Acr₂Shows the fluorescence spectrum measured by adding polydA, which is a single-stranded DNA.
FIG. 8 is a schematic diagram showing the fluorescence mechanism of the fluorescent reagent of the present invention.

Claims (17)

A fluorescent reagent capable of selectively detecting a double-stranded nucleic acid containing a fluorescent dye group and an intercalating group,
By contacting the double-stranded nucleic acid, the intercalating group in the fluorescent reagent molecule intercalates between the base pairs of the double-stranded nucleic acid, and the fluorescent dye group and the intercalating group in the fluorescent reagent molecule The quenching due to stacking is eliminated and the fluorescence intensity is enhanced, whereby double-stranded nucleic acids can be selectively detected.
A fluorescent reagent for selectively detecting a double-stranded nucleic acid containing a fluorescent dye group and an intercalating group.   The fluorescent reagent according to claim 1, wherein the fluorescent reagent is a peptide comprising an α-amino acid residue containing an α-amino acid residue substituted with a fluorescent dye group and an α-amino acid residue substituted with an intercalating group.   The fluorescent reagent according to claim 2, wherein an α-amino acid residue substituted with a fluorescent dye group and an α-amino acid residue substituted with an intercalating group are present every 2 to 10 α-amino acid residues.   The fluorescent reagent according to claim 3, wherein an α-amino acid residue substituted with a fluorescent dye group and an α-amino acid residue substituted with an intercalating group are present every two α-amino acid residues.   The fluorescent reagent according to any one of claims 2 to 4, wherein the total number of α-amino acid residues is 4 to 40.   The fluorescent reagent according to claim 5, wherein the total number of α-amino acid residues is 4 to 25.   The fluorescent reagent according to any one of claims 1 to 6, wherein the fluorescent dye group does not have a function as an intercalator. The fluorescent reagent according to any one of claims 1 to 7, wherein the fluorescent reagent is a peptide represented by the following formula I, a derivative thereof substituted at its N-terminal or C-terminal, or a salt thereof.

(Wherein X represents an intercalating group or fluorescent dye group, Y represents a divalent group constituting an α-amino acid, Z represents a monovalent group constituting an α-amino acid, and n represents 1 Represents an integer of 8 to 8. However, each X in the formula I may be different from or the same fluorescent dye group or intercalating group, and in the formula I, always an intercalating group and a fluorescent dye group. Both are present, and each Y and Z in Formula I may be different or the same.)   In the formula I, Y represents —AW— (A represents a linear or branched alkylene group having 1 to 6 carbon atoms, and W represents a single bond, NH, CO, CONH, NHCO, O, S, or HNC (NH) NH. The fluorescent reagent according to claim 8.   The fluorescent reagent according to claim 8 or 9, wherein in formula I, Z is -A-W-H (A and W are as defined in claim 9).   A group having an acridine skeleton which may have a substituent, an intercalate group, a group having an anthraquinone skeleton which may have a substituent, a group having an anthracene skeleton which may have a substituent, A group having a naphthalene imide skeleton which may have a substituent, a group having a naphthalene diimide skeleton which may have a substituent or a group having a pyrene skeleton which may have a substituent. The fluorescent reagent according to any one of 1 to 10.   The fluorescent dye group may have a substituent having a xanthene skeleton, a group having an indole skeleton optionally having a substituent, or a boron-containing and nitrogen indacene skeleton optionally having a substituent. The fluorescent reagent according to claim 1, wherein the fluorescent reagent is a group having The fluorescent reagent according to any one of claims 1 to 12, wherein the fluorescent reagent is a peptide represented by the following formula II, a derivative thereof substituted at its N-terminal or C-terminal, or a salt thereof.

A method for selectively detecting a double-stranded nucleic acid in a sample, comprising:
A fluorescent reagent according to any one of claims 1 to 13 is brought into contact with a specimen suspected of containing a double-stranded nucleic acid, and the double-stranded nucleic acid in the specimen is selectively detected by increasing the fluorescence intensity of the fluorescent reagent. how to.   The method according to claim 14, wherein the specimen is a DNA chip or a DNA microarray. A peptide represented by the following formula I, a derivative thereof substituted at the N-terminus or C-terminus, or a salt thereof.

(Wherein X represents an intercalating group or fluorescent dye group, Y represents a divalent group constituting an α-amino acid, Z represents a monovalent group constituting an α-amino acid, and n represents 1 Represents an integer of 8 to 8. However, each X in the formula I may be different from or the same fluorescent dye group or intercalating group, and in the formula I, always an intercalating group and a fluorescent dye group. Both are present, and each Y and Z in formula I may be different or the same).   The peptide according to claim 16, a derivative thereof substituted at the N-terminus or C-terminus thereof, or a salt thereof, wherein the fluorescent dye group does not have an intercalator function.

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