JPWO2005001086A1 - Immobilized mRNA-puromycin conjugate and uses thereof - Google Patents

Abstract

An object of the present invention is to provide an immobilized mRNA-puromycin conjugate, an mRNA bead or mRNA chip containing the conjugate, a protein chip prepared from the mRNA chip, a diagnostic kit using the mRNA bead or mRNA chip, mRNA or protein Solid phase immobilization method, protein solid phase synthesis method, method of analyzing interaction between protein and molecule using the immobilized mRNA-puromycin conjugate, and the like.

Description

  The present invention relates to an immobilized mRNA-puromycin conjugate, an immobilized mRNA / DNA-puromycin-protein conjugate, and uses thereof. More specifically, the present invention relates to an immobilized mRNA-puromycin conjugate, an mRNA bead or mRNA chip containing the conjugate, a protein chip prepared from the mRNA chip, a diagnostic kit using the mRNA bead or mRNA chip, and immobilization. MRNA / DNA-puromycin-protein conjugate, solid phase immobilization method of mRNA or protein, solid phase synthesis of protein, and interaction between protein and molecule using the immobilized mRNA-puromycin conjugate It relates to methods.

In the recent field of genomic science, research themes have shifted from "structural analysis" to clarify gene sequences to "functional analysis" by gene expression products. Basically, the function of a gene is realized because it is an expression product such as a protein. Therefore, protein analysis is essential for functional analysis of genes. Protein functional analysis is performed, for example, through biochemical functional analysis based on analysis of protein-protein interaction, protein-nucleic acid interaction, and the like.
As a method for analyzing protein-protein interaction, yeast two-hybrid method (Chien, CT, et al., Proc. Natl. Acad. Sci. USA, 88, 9578-9582 (1991)), phage display method (Smith, GP, Science, 228, pp. 1315-1317 (1985)), GST-fusion protein pull-down method, co-immunoprecipitation method and the like are known. Methods for analyzing protein-nucleic acid interactions include electrophoretic mobility shift assay (Revzin, A., et al., Anal. Biochem., 153, 172 (1986)), DNase I footprint method (Calas, D. et al. , Et al., Nucleic Acids Res., 5, 3157 (1978)), methylation buffer method and the like are known.
In addition, an in vitro virus method (see Nemoto et al., FEBS Lett. 414, 405 (1997); Tabuchi et al., FEBS Lett. 508, 309 (2001), etc.) using the specific properties of puromycin is used. A method for analyzing protein interaction has also been developed (see WO01 / 016600).
On the other hand, a DNA chip obtained by synthesizing DNA on a glass substrate or a protein chip in which proteins are arranged on a substrate is extremely important as a genome analysis tool or a gene function analysis tool. In addition, a protein in which an antibody-containing protein is immobilized on a substrate without losing its function is expected to be used in a wide range as a sensor chip in the future. However, proteins are unstable compared to DNA, and thus cannot be stored for a long period of time.

に示される化合物で、翻訳系でタンパク質の合成が行われた際に、合成されたタンパク質のＣ末端に結合する能力を有する。本明細書中、「ピューロマイシン様化合物」とは、その３’末端がアミノアシルｔＲＮＡに化学構造骨格が類似し、翻訳系でタンパク質の合成が行われた際に、合成されたタンパク質のＣ末端に結合する能力を有する化合物をいう。
ピューロマイシン様化合物としては、３’−Ｎ−アミノアシルピューロマイシンアミノヌクレオシド（３’−Ｎ−Ａｍｉｎｏａｃｙｌｐｕｒｏｍｙｃｉｎ ａｍｉｎｏｎｕｃｌｅｏｓｉｄｅ、ＰＡＮＳ−アミノ酸）、例えば、アミノ酸部がグリシンのＰＡＮＳ−Ｇｌｙ、アミノ酸部がバリンのＰＡＮＳ−Ｖａｌ、アミノ酸部がアラニンのＰＡＮＳ−Ａｌａ、その他、アミノ酸部が全ての各アミノ酸に対応するＰＡＮＳ−アミノ酸化合物が挙げられる。また、３’−アミノアデノシンのアミノ基とアミノ酸のカルボキシル基が脱水縮合して形成されるアミド結合で連結した３’−Ｎ−アミノアシルアデノシンアミノヌクレオシド（３’−Ａｍｉｎｏａｃｙｌａｄｅｎｏｓｉｎｅ ａｍｉｎｏｎｕｃｌｅｏｓｉｄｅ、ＡＡＮＳ−アミノ酸）、たとえば、アミノ酸部がグリシンのＡＡＮＳ−Ｇｌｙ、アミノ酸部がバリンのＡＡＮＳ−Ｖａｌ、アミノ酸部がアラニンのＡＡＮＳ−Ａｌａ、その他、アミノ酸部が全アミノ酸の各アミノ酸に対応するＡＡＮＳ−アミノ酸化合物を使用できる。また、ヌクレオシドあるいはヌクレオシドとアミノ酸のエステル結合したものなども使用できる。なお、上記ピューロマイシン以外に好ましく用いられるピューロマイシン様化合物は、リボシチジルピューロマイシン（ｒＣｐＰｕｒ）、デオキシジルピューロマイシン（ｄＣｐＰｕｒ）、デオキシウリジルピューロマイシン（ｄＵｐＰｕｒ）などであり、下記にその化学構造式を示す。

本発明のｍＲＮＡ−ＰＭ連結体においては、ｍＲＮＡとピューロマイシン又はピューロマイシン様化合物（以下、単に「ピューロマイシン」と称する）は、通常、スペーサーを介して連結される。また、この連結体は、通常、このスペーサーに設けられた固相結合部位を介して固相に固定化される。スペーサーは、主として、ピューロマイシンをリボソームのＡサイトと呼ばれる部位に効率良く取り込ませるために用いられる。したがって、スペーサーとしては、そのような性質を有する限り特に限定されないが、柔軟性があり、親水性で、側鎖の少ない単純な構造を有する骨格を有するものが好ましい。具体的には、ここで用いられるスペーサーとして、これらに限定されないが、ポリヌクレオチド（ＤＮＡ含む）、ポリエチレンなどのポリアルキレン、ポリエチレングリコールなどのポリアルキレングリコール、ペプチド核酸（ＰＮＡ）、ポリスチレン等の直鎖状物質又はこれらの組合せを主骨格として含むものが好ましく用いられる。上記直鎖上物質を組み合わせて用いる際は、適宜、それらを適当な連結基（−ＮＨ−、−ＣＯ−、−Ｏ−、−ＮＨＣＯ−、−ＣＯＮＨ−、−ＮＨＮＨ−、−（ＣＨ_２）_ｎ−［ｎは例えば１〜１０、好ましくは１〜３］、−Ｓ−、−ＳＯ−など）で化学的に連結することができる。
ｍＲＮＡとスペーサーとの連結は、公知の手法を用いて直接的又は間接的に、化学的又は物理的に行うことができる。例えば、ＤＮＡをスペーサーとして用いる場合は、ｍＲＮＡの３’末端にそのＤＮＡスペーサーの末端と相補的な配列を設けておくことにより、両者を連結することができる。また、スペーサーとピューロマイシンを連結する場合は、通常、公知の化学的手法によって連結される。
なお、タンパク質を合成した後に、ｍＲＮＡ−ＰＭ−タンパク質の複合体を固相から切り離す必要がある場合は、スペーサー中に切断可能部位を設けると好ましい。ＤＮＡをスペーサーの一部に用いた場合は、そのような切断可能部位として、ＤＮＡ鎖中に制限酵素認識部位を設けることができる。このような制限酵素認識部位をもつスペーサーを用いた場合は、タンパク質合成後など所望の時に、制限酵素（例えば、ＡｌｕＩ、ＢａｍＨＩ、ＥｃｏＲＩ、ＨｉｎｄＩＩ、ＨｉｎｄＩＩＩ、ＰｖｕＩＩなど）を投入することによって、ｍＲＮＡ−ＰＭ−タンパク質の複合体を固相から切り離すことができる。制限酵素認識部位とその部位を切断する酵素の組合せは公知である（Ｎｅｗ Ｅｎｇｌａｎｄ ＢｉｏＬａｂｓ ２０００・０１ Ｃａｔａｌｏｇ ＆ Ｔｅｃｈｎｉｃａｌ ｒｅｆｅｒｅｎｃｅ等参照）。
なお、本発明のｍＲＮＡ−ＰＭ連結体には、必要に応じて標識物質を結合させることによって標識することができる。そのような標識物質は、蛍光性物質、放射性標識物質などから適宜選択される。蛍光物質としては、フリーの官能基（例えば活性エステルに変換可能なカルボキシル基、ホスホアミダイドに変換可能な水酸基、あるいはアミノ基など）を持ち、スペーサー又はピューロマイシン又はピューロマイシン様化合物に連結可能な種々の蛍光色素を用いることができる。適当な標識物質としては、例えばフルオレスセインイソチオシアネート、フィコビリタンパク、希土類金属キレート、ダンシルクロライド若しくはテトラメチルローダミンイソチオシアネート等の蛍光物質；^３Ｈ、^１４Ｃ、^１２５Ｉ若しくは^１３１Ｉ等の放射性同位体などが挙げられる。
２．ｍＲＮＡの固相固定化方法
本発明の別の態様によれば、上記のｍＲＮＡ−ＰＭ連結体を用いて、ｍＲＮＡを固相に固定化する方法が提供される。すなわち、本発明のｍＲＮＡの固相固定化法は、
（ａ）固相結合部位を設けたスペーサーを介して、ｍＲＮＡとピューロマイシンを連結して、ｍＲＮＡ−ＰＭ連結体を調製する工程、及び、
（ｂ）該スペーサーの固相結合部位を固相に結合させることによって、該ｍＲＮＡ−ＰＭ連結体を固相に固定する工程
を含む。
ｍＲＮＡ−ＰＭ連結体が固定される固相は特に限定されず、その連結体が使用される目的に応じて適宜選択される。本発明で用いられる固相としては、生体分子を固定する担体となるものを用いることができ、例えば、スチレンビーズ、ガラスビーズ、アガロースビーズ、セファロースビーズ、磁性体ビーズ等のビーズ；ガラス基板、シリコン（石英）基板、プラスチック基板、金属基板（例えば、金箔基板）等の基板；ガラス容器、プラスチック容器等の容器；ニトロセルロース、ポリビニリデンフルオリド（ＰＶＤＦ）等の材料からなるメンブレンなどが挙げられる。なお、本明細書では、上記ｍＲＮＡ−ＰＭ連結体がビーズに固定されたものを「ｍＲＮＡビーズ」という。本発明の好ましい態様においては、ｍＲＮＡとピューロマイシン又はピューロマイシン様化合物との連結体がビーズに固定してなるｍＲＮＡビーズを提供する。
本発明のｍＲＮＡ−ＰＭ連結体は、ｍＲＮＡが翻訳系と接触する際に、その翻訳の障害とならないように固相に固定されれば、その固定化手段は特に限定されない。通常は、ｍＲＮＡとＰＭを連結するスペーサーに固相結合部位を設け、その固相結合部位を、固相に結合させた「固相結合部位認識部位」を介して、ｍＲＮＡ−ＰＭ連結体を固相に固定する。固相結合部位は、ｍＲＮＡ−ＰＭ連結体を所望の固相に結合し得るものであれば特に限定されない。例えば、このような固相結合部位として、特定のポリペプチドに特異的に結合する分子（例えば、リガンド、抗体など）が用いられ、この場合は、固相表面には固相結合部位認識部位として、その分子と結合する特定のポリペプチドを結合させておく。固相結合部位／固相結合部位認識部位の組合せの例としては、例えば、アビジン及びストレプトアビジン等のビオチン結合タンパク質／ビオチン、マルトース結合タンパク質／マルトース、Ｇタンパク質／グアニンヌクレオチド、ポリヒスチジンペプチド／ニッケルあるいはコバルト等の金属イオン、グルタチオン−Ｓ−トランスフェラーゼ／グルタチオン、ＤＮＡ結合タンパク質／ＤＮＡ、抗体／抗原分子（エピトープ）、カルモジュリン／カルモジュリン結合ペプチド、ＡＴＰ結合タンパク質／ＡＴＰ、あるいはエストラジオール受容体タンパク質／エストラジオールなどの、各種受容体タンパク質／そのリガンドなどが挙げられる。これらの中で、固相結合部位／固相結合部位認識部位の組合せとしては、アビジン及びストレプトアビジンなどのビオチン結合タンパク質、マルトース結合タンパク質／マルトース、ポリヒスチジンペプチド／ニッケルあるいはコバルト等の金属イオン、グルタチオン−Ｓ−トランスフェラーゼ／グルタチオン、抗体／抗原分子（エピトープ）などが好ましく、特にストレプトアビジン／ビオチンの組合せが最も好ましい。
上記タンパク質の固相表面への結合は、公知の方法を用いることができる。そのような公知の方法としては、例えば、タンニン酸、ホルマリン、グルタルアルデヒド、ピルビックアルデヒド、ビス−ジアゾ化ベンジゾン、トルエン−２，４−ジイソシアネート、アミノ基、カルボキシル基、又は水酸基あるいはアミノ基などを利用する方法を挙げることができる（Ｐ．Ｍ．Ａｂｄｅｌｌａ，Ｐ．Ｋ．Ｓｍｉｔｈ，Ｇ．Ｐ．Ｒｏｙｅｒ，Ａ Ｎｅｗ Ｃｌｅａｖａｂｌｅ Ｒｅａｇｅｎｔ ｆｏｒ Ｃｒｏｓｓ−Ｌｉｎｋｉｎｇ ａｎｄ Ｒｅｖｅｒｓｉｂｌｅ Ｉｍｍｏｂｉｌｉｚａｔｉｏｎ ｏｆ Ｐｒｏｔｅｉｎｓ，Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍｕｎ．，８７，７３４（１９７９）等参照）。
なお、上記組合せは、固相結合部位と固相結合部位認識部位とを逆転させて用いることもできる。上記の固定化手段は、２つの相互に親和性を有する物質を利用した固定化方法であるが、固相がスチレンビーズ、スチレン基板などのプラスチック材料であれば、必要に応じて、公知の手法を用いてスペーサーの一部を直接それらの固相に共有結合させることもできる（Ｑｉａｇｅｎ社、ＬｉｑｕｉＣｈｉｐ Ａｐｐｌｉｃａｔｉｏｎｓ Ｈａｎｄｂｏｏｋ等参照）。なお、本発明においては、固定手段については上記の方法に限定されることなく、当業者に公知である如何なる固定手段をも利用することができる。
３．タンパク質の固相固定化方法及びタンパク質の固相合成方法
本発明の別の態様によれば、タンパク質の固相固定化又は固相合成法であって、上記（ｂ）工程の後に、（ｃ）該ｍＲＮＡ−ＰＭ連結体と翻訳系とを接触させることにより（例えば、該連結体に翻訳系を投入、あるいは該連結体を翻訳系に投入）、タンパク質を合成する工程を含む、タンパク質の固相固定化又は合成法が提供される。工程（ｂ）において、ｍＲＮＡ−ＰＭ連結体が固相に固定されているので、この連結体を翻訳系に投入した際に、前述のｉｎ ｖｉｔｒｏ ｖｉｒｕｓ法の対応づけ技術を利用して、合成されたタンパク質がピューロマイシンを介して固相に固定化されるのである。
上記（ｃ）工程では、ｍＲＮＡ−ＰＭ連結体を翻訳系と接触させることによって、タンパク質の合成を行う。ここで用いることができる翻訳系としては、無細胞翻訳系又は生細胞などが挙げられる。無細胞翻訳系としては、原核又は真核生物の抽出物により構成される無細胞翻訳系、例えば大腸菌、ウサギ網状赤血球、小麦胚芽抽出物などが使用できる（Ｌａｍｆｒｏｍ Ｈ，Ｇｒｕｎｂｅｒｇ−Ｍａｎａｇｏ Ｍ．Ａｍｂｉｇｕｉｔｉｅｓ ｏｆ ｔｒａｎｓｌａｔｉｏｎ ｏｆ ｐｏｌｙ Ｕ ｉｎ ｔｈｅ ｒａｂｂｉｔ ｒｅｔｉｃｕｌｏｃｙｔｅ ｓｙｓｔｅｍ．Ｂｉｏｃｈｅｍ Ｂｉｏｐｈｙｓ Ｒｅｓ Ｃｏｍｍｕｎ．１９６７ ２７（１）：１−６等参照）。生細胞翻訳系としては、原核又は真核生物、例えば大腸菌の細胞などが使用できる。本発明においては、取り扱いの容易さから、無細胞系を使用することが好ましい。
ここで、図１に基づいて、本発明のｍＲＮＡ−ピューロマイシン連結体を用いたタンパク質の固定化について簡単に説明する。図１ａは保存時の固定化ｍＲＮＡ−ＰＭ連結体を示す図、図１ｂは無細胞翻訳系を投入し、タンパク質が合成されている状態を示す図、図１ｃはタンパク質の合成が終了した状態を示す図である。図１ａに示すように、固定化ｍＲＮＡ−ピューロマイシン連結体１は、ｍＲＮＡ１ａとピューロマイシン１ｂが、ＤＮＡスペーサー１ｃを介して連結されたもので、固相結合部位１ｄを介して固相２に結合されている。固相２は、後に、無細胞翻訳系を投入することを考慮して容器の形状をしている。図１ｂに示すように、固定化ｍＲＮＡ−ピューロマイシン連結体１に、無細胞翻訳系３を投入すると、ｍＲＮＡと無細胞翻訳系を利用した翻訳反応によって、そのｍＲＮＡの核酸配列に対応するタンパク質４が合成される。翻訳終了後は、不要な無細胞翻訳系３の成分を除去し、タンパク質４がピューロマイシン１ｂに結合した、ｍＲＮＡ−ピューロマイシン−タンパク質複合体が、固相２に固定された状態で形成される。なお、このようなｍＲＮＡ−ピューロマイシン−タンパク質複合体（連結体）に、例えば、標的物質を接触させることによって、合成されたタンパク質と結合し得る標的物質をスクリーニングすることができる。
４．ｍＲＮＡチップ及びプロテインチップ
本発明の別の態様によれば、上記した固定化ｍＲＮＡ−ピューロマイシン連結体を複数含むｍＲＮＡチップ（ｍＲＮＡマイクロアレイ）に関する。即ち本発明は、本発明の固定化ｍＲＮＡ−ピューロマイシン連結体をマイクロアレイ用基板に固定してなる、ｍＲＮＡチップを提供する。このｍＲＮＡチップは、上述したｍＲＮＡ−ＰＭ連結体を複数基板上に固定化したものである。なお、上記「複数」とは、特にその上限の値は制限されるものではないが、通常マイクロアレイとして用いられるチップ上に固定可能な実用的な数を指す。このｍＲＮＡチップを翻訳系に投入することにより、あるいは、翻訳系をｍＲＮＡチップに投入することにより、上述のタンパク質合成がチップ上で起こり、各タンパク質が固相に結合した、いわゆるプロテインチップが作製される。このようにして作製されるプロテインチップもまた、本発明に含まれる。
本発明のｍＲＮＡチップにおいては、機能既知のタンパク質をコードするｍＲＮＡ複数を、ｍＲＮＡ−ＰＭ連結体として固相に固定してもよいし、機能未知のタンパク質をコードするｍＲＮＡ複数をｍＲＮＡ−ピューロマイシン連結体として固相に固定してもよい。例えば、疾病に関与する機能既知のタンパク質をコードするｍＲＮＡを複数チップに固定する場合は、例えば、疾病の診断用ｍＲＮＡチップ、タンパク質相互作用解析用ｍＲＮＡチップ等とすることができる。診断用チップとして用いる場合は、ある特定の疾病の診断マーカーと結合するタンパク質をコードするｍＲＮＡをそれぞれプレートの所定の位置に固定しておく。そして、診断を行う直前にこのプレートに無細胞翻訳系を投入して、プレート上の所定の位置に所望の診断マーカーと結合するタンパク質を合成し、固定する。このようにすれば、診断の直前にプロテインチップを作製することができる。プロテインチップは、その保存上あるいは取り扱い上に問題がある。不安定なプロテインの代わりに安定なｍＲＮＡの形でチップ化した点は、本発明の特徴の一つと言える。ゆえに、ｍＲＮＡの固相固定化及びタンパク質の合成・固定化以外の技術（例えば、使用するプレートの材料・サイズ、使用するプロテインの種類・配置、プロテインチップを用いたタンパク質の機能解析方法等）は、公知のプロテインチップの技術をそのまま利用することができる（Ｋｕｋａｒ Ｔ，Ｅｃｋｅｎｒｏｄｅ Ｓ，Ｇｕ Ｙ，Ｌｉａｎ Ｗ，Ｍｅｇｇｉｎｓｏｎ Ｍ，Ｓｈｅ ＪＸ，Ｗｕ Ｄ．Ｐｒｏｔｅｉｎ ｍｉｃｒｏａｒｒａｙｓ ｔｏ ｄｅｔｅｃｔ ｐｒｏｔｅｉｎ−ｐｒｏｔｅｉｎ ｉｎｔｅｒａｃｔｉｏｎｓ ｕｓｉｎｇ ｒｅｄ ａｎｄ ｇｒｅｅｎ ｆｌｕｏｒｅｓｃｅｎｔ ｐｒｏｔｅｉｎｓ．Ａｎａｌ Ｂｉｏｃｈｅｍ．２００２；３０６（１）：５０−４等参照）。なお、上記ｍＲＮＡビーズ又は上記ｍＲＮＡチップと無細胞翻訳系を含む診断キット、あるいは、タンパク質相互作用解析用キットも本発明の範囲内である。このようなｍＲＮＡチップを用いることによって、タンパク質−タンパク質相互作用、ＤＮＡ−タンパク質相互作用、リガンドの探索、疾病マーカーの探索、疾病の診断、薬効評価、薬物動態の評価などに利用することができる。
また、本発明の固定化ｍＲＮＡ−ピューロマイシン連結体を翻訳系と接触させることによって合成されるタンパク質が、上記連結体へ付加された構造からなる連結体（「固定化ｍＲＮＡ−ピューロマイシン−タンパク質連結体」と記載する）も、本発明に含まれる。即ち本発明は、本発明の「固定化ｍＲＮＡ−ピューロマイシン連結体」を翻訳系へ供して合成される該ｍＲＮＡの翻訳産物のタンパク質（ポリペプチド）が、該連結体におけるピューロマイシン又はピューロマイシン様化合物を介して連結してなる、固定化ｍＲＮＡ−ピューロマイシン−タンパク質連結体に関する。該連結体は、例えば、後述の「タンパク質と標的分子との相互作用を解析する方法」に好適に用いることができる。また上記連結体の一つの態様としては、上述のように、固定化ｍＲＮＡ−ピューロマイシン−タンパク質連結体がマイクロアレイ用基板へ固定された構造のプロテインチップを例示することができる。
また本発明の好ましい態様においては、上記「固定化ｍＲＮＡ−ピューロマイシン−タンパク質連結体」を、逆転写反応系へ供する（逆転写反応系と接触させる）ことにより、該ｍＲＮＡの逆転写産物である相補的ＤＮＡ（該ｍＲＮＡとハイブリダイズするＤＮＡ）を合成することが可能である。例えば、本発明の上記スペーサーとして、ｍＲＮＡの３’末端配列と相補的な配列を含むＤＮＡスペーサーを用いる場合、該相補的な配列が該ｍＲＮＡの逆転写反応においてプライマーとして機能することが期待される。即ち、該ｍＲＮＡ−ピューロマイシン−タンパク質連結体を逆転写反応系へ供することにより、該プライマーを合成起点とするＤＮＡ合成反応が開始され、該ｍＲＮＡと相補的な配列からなるＤＮＡが合成される。このようにして合成されるＤＮＡを含む連結体（「ＤＮＡ−ピューロマイシン−タンパク質連結体」と記載する）もまた、本発明に含まれる。即ち本発明は、本発明の「固定化ｍＲＮＡ−ピューロマイシン−タンパク質連結体」を、逆転写反応系へ供して合成される該ｍＲＮＡの相補的ＤＮＡが、該連結体と結合してなる、固定化ＤＮＡ−ピューロマイシン−タンパク質連結体を提供する。
本発明の上記連結体においてピューロマイシンと連結される核酸分子は、通常、ｍＲＮＡと該ｍＲＮＡの相補的ＤＮＡとの二本鎖核酸分子であるが、当該核酸分子におけるｍＲＮＡは、その後のヌクレアーゼ反応等によって消化されていてもよい。即ち、一本鎖の（ｍＲＮＡと相補的）ＤＮＡ分子がピューロマイシンと連結された構造からなる「固定化ＤＮＡ−ピューロマイシン−タンパク質連結体」もまた、本発明の連結体の一つの態様である。さらに、本発明の上記連結体において、ピューロマイシンと連結される核酸分子は、該ＤＮＡと相補性を有するＤＮＡからなる二本鎖ＤＮＡであってもよい。
なお、本発明において「逆転写反応系」とは、ｍＲＮＡを鋳型としてＤＮＡを合成する所謂「逆転写」を司る反応系をいい、当該反応系は通常、逆転写酵素を含有する。本発明の逆転写反応は、当業者においては、適宜実施することが可能である。より具体的には、後述の実施例に記載の方法により行うことができる。
５．タンパク質と分子との相互作用を解析する方法
本発明の別の態様によれば、上記したｍＲＮＡ−ＰＭ連結体を用いたタンパク質と分子との相互作用を解析する方法が提供される。この解析方法は、
（ａ）一以上の本発明の固定化ｍＲＮＡ−ピューロマイシン連結体を、翻訳系に投入し、固相上でタンパク質を合成する工程；
（ｂ）工程（ａ）において合成されたタンパク質と一以上の標的物質とを接触させる工程；及び
（ｃ）該タンパク質と該標的物質とが相互作用しているか否かを測定する工程を含む。
この解析方法は、例えば、（ｉ）配列既知のタンパク質に作用する物質をスクリーニングする場合、（ｉｉ）ある特定の物質（例えば、リガンド）が結合する配列未知のタンパク質をスクリーニングする場合等に用いることができる。例えば、（ｉ）の場合は、配列既知のタンパク質（例えばオーファンレセプタータンパク質）をコードする核酸配列を有するｍＲＮＡとピューロマイシンとの連結体を複数用意しておき（すなわち、複数のオーファンレセプタータンパク質に対応するｍＲＮＡをそれぞれ有するｍＲＮＡ−ＰＭ連結体を複数用意）、これを翻訳系に投入する。すると、各ｍＲＮＡ−ＰＭ連結体のｍＲＮＡから複数のオーファンレセプタータンパク質が合成される。各オーファンレセプタータンパク質は、固相に固定されたｍＲＮＡ−ＰＭ連結体のピューロマイシンにＣ末端が結合することによって固定される。必要に応じて、不要な成分を洗浄除去し、これに標的物質及びバッファー等を加えて、標的物質をオーファンレセプタータンパク質に結合させることによって、結合実験を行う。（ｉｉ）の場合は、例えば、ある遺伝子ライブラリーから複数のｍＲＮＡを取得し、複数のｍＲＮＡとピューロマイシンとの連結体を作成し、固相に固定する。以下、同様にタンパク質の合成を行い、標的物質をそのタンパク質に接触させて結合実験を行う。
上記工程（ｂ）においては、工程（ａ）において合成されたタンパク質と一以上の標的物質とを接触させる。ここで用いられる「標的物質」とは、本発明において合成されるタンパク質と相互作用するか否か調べるための物質を意味し、具体的にはタンパク質、核酸、糖鎖、低分子化合物などが挙げられる。
タンパク質としては、特に制限はなく、タンパク質の全長であっても結合活性部位を含む部分ペプチドでもよい。またアミノ酸配列、及びその機能が既知のタンパク質でも、未知のタンパク質でもよい。これらは、合成されたペプチド鎖、生体より精製されたタンパク質、あるいはｃＤＮＡライブラリー等から適当な翻訳系を用いて翻訳し、精製したタンパク質等でも標的分子として用いることができる。合成されたペプチド鎖はこれに糖鎖が結合した糖タンパク質であってもよい。これらのうち好ましくはアミノ酸配列が既知の精製されたタンパク質か、あるいはｃＤＮＡライブラリー等から適当な方法を用いて翻訳、精製されたタンパク質を用いることができる。
核酸としては、特に制限されることはなく、ＤＮＡあるいはＲＮＡも用いることができる。また、塩基配列あるいは機能が既知の核酸でも、未知の核酸でもよい。好ましくは、タンパク質に結合能力を有する核酸としての機能、及び塩基配列が既知のものか、あるいはゲノムライブラリー等から制限酵素等を用いて切断単離してきたものを用いることができる。
糖鎖としては、特に制限はなく、その糖配列あるいは機能が、既知の糖鎖でも未知の糖鎖でもよい。好ましくは、既に分離解析され、糖配列あるいは機能が既知の糖鎖が用いられる。
低分子化合物としては、特に制限されず、機能が未知のものでも、あるいはタンパク質に結合する能力が既に知られているものでも用いることができる。
なお、これら標的物質とタンパク質との「相互作用」とは、通常は、タンパク質と標的分子間の共有結合、疎水結合、水素結合、ファンデルワールス結合、及び静電力による結合のうち少なくとも１つから生じる分子間に働く力による作用を示すが、この用語は最も広義に解釈すべきであり、いかなる意味においても限定的に解釈してはならない。共有結合としては、配位結合、双極子結合を含有する。また静電力による結合とは、静電結合の他、電気的反発も含有する。また、上記作用の結果生じる結合反応、合成反応、分解反応も相互作用に含有される。相互作用の具体例としては、抗原と抗体間の結合及び解離、タンパク質レセプターとリガンドの間の結合及び解離、接着分子と相手方分子の間の結合及び解離、酵素と基質の間の結合及び解離、核酸とそれに結合するタンパク質の間の結合及び解離、情報伝達系におけるタンパク質同士の間の結合と解離、糖タンパク質とタンパク質との間の結合及び解離、あるいは糖鎖とタンパク質との間の結合及び解離が挙げられる。
ここで用いられる標的物質は、必要に応じて標識物質により標識して用いることができる。必要に応じて標識物質を結合させることによって標識することができる。そのような標識物質は、蛍光性物質、放射性標識物質などから適宜選択される。蛍光物質としては、フリーの官能基（例えば活性エステルに変換可能なカルボキシル基、ホスホアミダイドに変換可能な水酸基、あるいはアミノ基など）を持ち、標的物質に連結可能な種々の蛍光色素を用いることができる。適当な標識物質としては、例えばフルオレスセインイソチオシアネート、フィコビリタンパク、希土類金属キレート、ダンシルクロライド若しくはテトラメチルローダミンイソチオシアネート等の蛍光物質；^３Ｈ、^１４Ｃ、^１２５Ｉ若しくは^１３１Ｉ等の放射性同位体などが挙げられる。これらの標識物質は、標的物質と固定化タンパク質との間の相互作用に基づいて発生される信号の変化の測定又は解析方法に適したものが適宜用いられる。上記標識物質の標的物質への結合は、公知の手法に基づいて行うことができる。
次いで、本解析方法によれば、工程（ｃ）において、該タンパク質と該標的物質とが相互作用しているか否かを測定する。該タンパク質と該標的物質とが相互作用しているか否かの測定は、両分子間の相互作用に基づいて発生される信号の変化を測定、検出することにより行う。そのような測定手法としては、例えば、表面プラズモン共鳴法（Ｃｕｌｌｅｎ，Ｄ．Ｃ．，ｅｔ ａｌ．，Ｂｉｏｓｅｎｓｏｒｓ，３（４），２１１−２２５（１９８７−８８））、エバネッセント場分子イメージング法（Ｆｕｎａｔｓｕ，Ｔ．，ｅｔ ａｌ．，Ｎａｔｕｒｅ，３７４，５５５−５５９（１９９５））、蛍光イメージングアナライズ法、固相酵素免疫検定法（Ｅｎｚｙｍｅ Ｌｉｎｋｅｄ Ｉｍｍｕｎｏｓｏｒｂｅｎｔ Ａｓｓａｙ（ＥＬＩＳＡ）：Ｃｒｏｗｔｈｅｒ，Ｊ．Ｒ．，Ｍｅｔｈｏｄｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ，４２（１９９５））、蛍光偏光解消法（Ｐｅｒｒａｎ，Ｊ．，ｅｔ ａｌ．，Ｊ．Ｐｈｙｓ．Ｒａｄ．，１，３９０−４０１（１９２６））、及び蛍光相関分光法（Ｆｌｕｏｒｅｓｃｅｎｃｅ Ｃｏｒｒｅｌａｔｉｏｎ Ｓｐｅｃｔｒｏｓｃｏｐｙ（ＦＣＳ）：Ｅｉｇｅｎ，Ｍ．，ｅｔ ａｌ．，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，９１，５７４０−５７４７（１９９４））等が挙げられる。
本発明の解析方法においては、必要に応じて、さらに、工程（ｃ）において相互作用していると判断されたタンパク質−標的物質結合体中の、タンパク質及び／又は標的物質を同定する。タンパク質の同定は、通常のアミノ酸配列シークエンサーで行うこともできるし、該タンパク質に結合しているｍＲＮＡからＤＮＡを逆転写し、得られたＤＮＡの塩基配列を解析することによって行うこともできる。標的物質の同定は、ＮＭＲ、ＩＲ、各種質量分析などによって行うことができる。なお、本発明のｍＲＮＡチップ及びプロテインチップを用いて、タンパク質−タンパク質間相互作用を解析する場合は、通常のプロテインチップ上のサンプル解析と同様に、飛行時間型質量分析計（ＭＡＬＤＩ−ＴＯＦ ＭＳ）を用いることができる。
また本発明の上記方法は、上記工程（ａ）に続いて、工程（ａ）において合成されるｍＲＮＡ−ピューロマイシン−タンパク質連結体を逆転写反応系へ供することにより、該連結体におけるｍＲＮＡと相補的なＤＮＡを含有する連結体（ＤＮＡ−ピューロマイシン−タンパク質連結体）を作製し、上述のその後の工程を実施させてもよい。即ち本発明の好ましい態様においては、以下の工程を含むタンパク質と分子との相互作用を解析する方法を提供する。
（ａ）一以上の、本発明のいずれかに記載の固定化ｍＲＮＡ−ピューロマイシン連結体と、翻訳系とを接触させて、固相上でタンパク質を合成する工程；
（ｂ）工程（ａ）において合成されたｍＲＮＡ−ピューロマイシン−タンパク質連結体と、逆転写反応系とを接触させて、ＤＮＡ−ピューロマイシン−タンパク質連結体を調製する工程；
（ｃ）工程（ｂ）において調製されたＤＮＡ−ピューロマイシン−タンパク質連結体と一以上の標的物質とを接触させる工程；及び
（ｄ）該連結体におけるタンパク質と該標的物質とが相互作用しているか否かを測定する工程；As described above, various gene function analysis techniques have been developed, but even now, development of tools for performing gene function analysis more efficiently and rapidly is desired. In particular, the development of an expression method and an immobilization method while maintaining the protein function stably is desired.
The present inventors have intensively studied to solve the above problems. First, the present inventors developed a tool that can easily determine the interaction between a protein having various amino acid sequences and a target substance. That is, the present inventors are a conjugate of a nucleic acid molecule (mRNA) encoding a test protein whose interaction with a target substance is to be determined, and puromycin that plays a role as a linking moiety when the protein is synthesized. Produced “an immobilized mRNA-puromainsin conjugate” having a structure immobilized on a solid phase.
It is possible to synthesize a protein on a solid phase by bringing the immobilized mRNA-puromainsin conjugate into contact with a translation system. The synthesized protein is immobilized on the solid phase via puromycin.
As a preferable embodiment of the above-mentioned immobilized mRNA-puromainsin conjugate, a microarray chip (mRNA chip) immobilized on a substrate such as a chip can be exemplified. A protein chip can be appropriately produced by bringing the chip into contact with a translation system. Usually, protein (protein) chips have problems in storage or handling because proteins are unstable. In a preferred embodiment of the present invention, it is possible to convert a chip into a protein chip by forming a chip in the form of a relatively stable mRNA instead of a protein, for example, by contacting with a translation system immediately before use. . That is, the present invention provides a protein interaction analysis tool that is immobilized on a solid phase in the form of a relatively stable mRNA instead of an unstable protein.
In addition, the “immobilized mRNA-puromycin-protein conjugate” in which the protein of the translation product of the mRNA synthesized by subjecting the immobilized mRNA-puromycin conjugate to a translation system is bound to the puromycin is a protein. It can be used for the analysis or screening of molecules that can interact with, and is very useful.
Furthermore, the present inventors provided the above-mentioned “immobilized mRNA-puromycin-protein conjugate” to a reverse transcription reaction system, whereby “immobilized DNA-puro” in which complementary DNAs of the reverse transcription product of the mRNA were linked. "Mycin-protein conjugate" was successfully produced. Since DNA is usually more stable than RNA, when contacting the conjugate with a test molecule in protein interaction analysis, the “immobilized DNA-puromycin-protein conjugate” is very Useful.
The present invention has been made to solve the above-mentioned problems of the prior art, and is prepared from an immobilized mRNA-puromycin conjugate, an mRNA bead or mRNA chip containing this conjugate, and an mRNA chip as shown below. Protein chip, diagnostic kit using mRNA beads or mRNA chip, immobilized DNA-puromycin-protein conjugate, solid phase immobilization method of mRNA or protein, solid phase synthesis method of protein, immobilized mRNA-puro A method for analyzing an interaction between a protein and a molecule using a mycin conjugate is provided. More specifically, the present invention provides:
[1] An immobilized mRNA-puromycin conjugate obtained by immobilizing a conjugate of mRNA and puromycin or a puromycin-like compound on a solid phase,
[2] The immobilized mRNA-puromycin conjugate according to [1], wherein the mRNA-puromycin conjugate is obtained by linking puromycin or a puromycin-like compound to the 3 ′ end of mRNA via a spacer.
[3] The immobilized mRNA-puromycin conjugate according to [1] or [2], wherein the mRNA-puromycin conjugate is bound to a solid phase via a solid phase binding site provided in the spacer. ,
[4] The immobilized mRNA-puromycin linkage according to any one of [1] to [3], wherein the spacer includes a polynucleotide, polyethylene, polyethylene glycol, polystyrene, peptide nucleic acid, or a combination thereof as a main skeleton. body,
[5] The solid phase is selected from styrene beads, glass beads, agarose beads, sepharose beads, magnetic beads, glass substrates, silicon substrates, plastic substrates, metal substrates, glass containers, plastic containers, and membranes. ] The immobilized mRNA-puromycin conjugate according to any one of [4] to [4],
[6] The translation product protein of the mRNA synthesized by subjecting the immobilized mRNA-puromycin conjugate according to any one of [1] to [5] to a translation system is a puromycin or a puro in the conjugate. An immobilized mRNA-puromycin-protein conjugate, linked via a mycin-like compound,
[7] Immobilization wherein complementary mRNA of the mRNA synthesized by subjecting the immobilized mRNA-puromycin-protein conjugate according to [6] to a reverse transcription reaction system is bound to the conjugate. DNA-puromycin-protein conjugate,
[8] An mRNA chip, wherein the immobilized mRNA-puromycin conjugate according to any one of [1] to [5] is immobilized on a microarray substrate,
[9] A protein chip produced using the mRNA chip according to [8],
[10] mRNA beads in which a conjugate of mRNA and puromycin or puromycin-like compound is immobilized on the beads,
[11] A protein interaction analysis kit comprising the mRNA chip according to [8] or the mRNA bead according to [10], and a cell-free translation system,
[12] (a) linking mRNA and puromycin via a spacer provided with a solid phase binding site to prepare an mRNA-puromycin conjugate, and
(B) fixing the mRNA-puromycin conjugate to a solid phase by binding the solid phase binding site of the spacer to the solid phase
A method for immobilizing mRNA on a solid phase, comprising:
[13] (a) A step of preparing an mRNA-puromycin conjugate by linking mRNA and puromycin via a spacer provided with a solid phase binding site;
(B) fixing the mRNA-puromycin conjugate to a solid phase by binding the solid phase binding site of the spacer to the solid phase; and
(C) A step of synthesizing a protein by bringing the mRNA-puromycin conjugate into contact with a translation system.
A method for immobilizing a protein on a solid phase, comprising:
[14] (a) A step of preparing an mRNA-puromycin conjugate by linking mRNA and puromycin via a spacer provided with a solid phase binding site;
(B) fixing the mRNA-puromycin conjugate to a solid phase by binding the solid phase binding site of the spacer to the solid phase; and
(C) A step of synthesizing a protein by bringing the mRNA-puromycin conjugate into contact with a translation system.
A method for synthesizing a protein in a solid phase, comprising
[15] A method for analyzing the interaction between a protein and a molecule,
(A) contacting one or more of the immobilized mRNA-puromycin conjugate according to any one of [1] to [5] with a translation system to synthesize a protein on a solid phase;
(B) contacting the protein synthesized in step (a) with one or more target substances; and
(C) a step of measuring whether or not the protein and the target substance interact;
Including the above analysis method,
[16] A method for analyzing the interaction between a protein and a molecule,
(A) contacting one or more of the immobilized mRNA-puromycin conjugate according to any one of [1] to [5] with a translation system to synthesize a protein on a solid phase;
(B) contacting the mRNA-puromycin-protein conjugate synthesized in step (a) with a reverse transcription reaction system to prepare a DNA-puromycin-protein conjugate;
(C) contacting the DNA-puromycin-protein conjugate prepared in step (b) with one or more target substances; and
(D) a step of measuring whether or not the protein in the conjugate and the target substance interact;
Including the above analysis method,
[17] The analysis method according to [15] or [16], further comprising the step of identifying a protein and / or target substance determined to be interacting,
Is provided.
Hereinafter, the present invention will be described in detail based on the embodiments.
1. Immobilized mRNA-puromycin conjugate
The first aspect of the present invention is an immobilized mRNA-puromycin conjugate (hereinafter referred to as “immobilized mRNA-PM conjugate”), which is obtained by immobilizing a conjugate of mRNA and puromycin or a puromycin-like compound to a solid phase. ").
The mRNA used in the present invention includes both those with unknown sequences and those with known sequences. That is, when searching for or quantifying a substance that binds to a protein with a known sequence using the mRNA-PM conjugate of the present invention, mRNA having a nucleic acid sequence encoding a protein with a known sequence is used. Conversely, when analyzing the function of a protein whose sequence is unknown using the mRNA-PM conjugate of the present invention, mRNA having a nucleic acid sequence encoding a protein whose sequence is unknown can be used. As used herein, mRNA is, for example, mRNA encoding various receptor proteins with known sequences, mRNA encoding various antibodies or fragments thereof, mRNA encoding various enzymes, and sequences transcribed from DNA in various gene libraries. It is selected from unknown mRNA, mRNA having a random sequence transcribed from DNA having a sequence synthesized randomly by organic synthesis, and the like.
In the mRNA-PM conjugate of the present invention, puromycin is usually linked to the 3 ′ end of mRNA via a spacer. Here, puromycin or puromycin-like compounds are hinges or linkages that link mRNA and translated proteins when synthesizing proteins by introducing mRNA-PM conjugates immobilized on a solid phase into the translation system. To act as a department. That is, it is known that when a translation system is brought into contact with puromycin bound to mRNA via a spacer, an in vitro virus virion in which the mRNA is bound to a protein translated via puromycin is known. (See Nemoto et al., FEBS Lett. 414, 405 (1997)). Puromycin has a chemical structural skeleton similar to aminoacyl tRNA at the 3 ′ end, and is represented by the following formula (I):

When the protein is synthesized in a translation system, it has the ability to bind to the C-terminus of the synthesized protein. In the present specification, a “puromycin-like compound” has a chemical structure skeleton similar to aminoacyl-tRNA at the 3 ′ end, and is synthesized at the C-terminus of the synthesized protein when the protein is synthesized in a translation system. A compound having the ability to bind.
Examples of puromycin-like compounds include 3'-N-aminoacylpuromycin aminonucleoside (3'-N-aminoacylpurinomycin amino acid, PANS-amino acid), for example, PANS-Gly having an amino acid part of glycine and PANS-Val having an amino acid part of valine. PANS-Ala whose amino acid part is alanine, and other PANS-amino acid compounds whose amino acid part corresponds to all amino acids. Also, 3′-N-aminoacyl adenosine aminonucleoside (3′-Aminoacylenenosine aminonucleotide, AANS-amino acid) linked by an amide bond formed by dehydration condensation of the amino group of 3′-aminoadenosine and the carboxyl group of amino acid, for example, AANS-Gly whose amino acid portion is glycine, AANS-Val whose amino acid portion is valine, AANS-Ala whose amino acid portion is alanine, and other AANS-amino acid compounds whose amino acid portions correspond to each amino acid of all amino acids can be used. Further, nucleosides or those obtained by ester bonding of nucleosides and amino acids can also be used. Puromycin-like compounds preferably used in addition to the above puromycin include ribocytidylpuromycin (rCpPur), deoxydylpuromycin (dCpPur), deoxyuridylpuromycin (dUpPur), and the chemical structure thereof is shown below. An expression is shown.

In the mRNA-PM conjugate of the present invention, mRNA and puromycin or puromycin-like compound (hereinafter simply referred to as “puromycin”) are usually linked via a spacer. Moreover, this coupling body is normally fix | immobilized by the solid phase through the solid-phase binding site provided in this spacer. The spacer is mainly used to efficiently incorporate puromycin into a site called A site of ribosome. Therefore, the spacer is not particularly limited as long as it has such properties, but a spacer having a skeleton having a simple structure with flexibility, hydrophilicity, and few side chains is preferable. Specifically, spacers used herein are not limited to these, but include polynucleotides (including DNA), polyalkylenes such as polyethylene, polyalkylene glycols such as polyethylene glycol, peptide nucleic acids (PNA), and linear chains such as polystyrene. A substance containing a substance or a combination thereof as a main skeleton is preferably used. When the above linear substances are used in combination, they are appropriately combined with an appropriate linking group (—NH—, —CO—, —O—, —NHCO—, —CONH—, —NHNH—, — (CH ₂ ) _n -[N is 1 to 10, preferably 1 to 3, for example, -S-, -SO-, etc.].
The linkage between mRNA and spacer can be performed directly or indirectly, chemically or physically using a known technique. For example, when DNA is used as a spacer, both can be linked by providing a sequence complementary to the end of the DNA spacer at the 3 ′ end of mRNA. In addition, when the spacer and puromycin are linked, they are usually linked by a known chemical method.
In addition, when it is necessary to separate the mRNA-PM-protein complex from the solid phase after the protein is synthesized, it is preferable to provide a cleavable site in the spacer. When DNA is used as a part of the spacer, a restriction enzyme recognition site can be provided in the DNA strand as such a cleavable site. When a spacer having such a restriction enzyme recognition site is used, by introducing a restriction enzyme (for example, AluI, BamHI, EcoRI, HindII, HindIII, PvuII, etc.) at a desired time such as after protein synthesis, mRNA- The PM-protein complex can be cleaved from the solid phase. A combination of a restriction enzyme recognition site and an enzyme that cleaves the site is known (see, for example, New England BioLabs 2000 · 01 Catalog & Technical reference).
The mRNA-PM conjugate of the present invention can be labeled by binding a labeling substance as necessary. Such a labeling substance is appropriately selected from fluorescent substances, radioactive labeling substances and the like. As fluorescent substances, there are various functional groups having free functional groups (for example, a carboxyl group that can be converted into an active ester, a hydroxyl group that can be converted into a phosphoramidide, or an amino group) and can be linked to a spacer or puromycin or puromycin-like compound. A fluorescent dye can be used. Suitable labeling substances include, for example, fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate, dansyl chloride or tetramethylrhodamine isothiocyanate; ³ H, ¹⁴ C, ¹²⁵ I or ¹³¹ And radioisotopes such as I.
2. Method for solid phase immobilization of mRNA
According to another aspect of the present invention, there is provided a method for immobilizing mRNA on a solid phase using the above mRNA-PM conjugate. That is, the solid phase immobilization method of mRNA of the present invention comprises:
(A) a step of preparing mRNA-PM conjugate by linking mRNA and puromycin via a spacer provided with a solid phase binding site; and
(B) fixing the mRNA-PM conjugate to the solid phase by binding the solid phase binding site of the spacer to the solid phase
including.
The solid phase on which the mRNA-PM conjugate is immobilized is not particularly limited, and is appropriately selected depending on the purpose for which the conjugate is used. As the solid phase used in the present invention, one that serves as a carrier for immobilizing biomolecules can be used. For example, beads such as styrene beads, glass beads, agarose beads, sepharose beads, magnetic beads; glass substrates, silicon Examples include (quartz) substrates, plastic substrates, metal substrates (for example, gold foil substrates) and the like; containers such as glass containers and plastic containers; membranes made of materials such as nitrocellulose and polyvinylidene fluoride (PVDF). In the present specification, the mRNA-PM conjugate immobilized on the beads is referred to as “mRNA beads”. In a preferred embodiment of the present invention, an mRNA bead is provided in which a conjugate of mRNA and puromycin or puromycin-like compound is immobilized on the bead.
The mRNA-PM conjugate of the present invention is not particularly limited as long as it is immobilized on a solid phase so that the mRNA does not interfere with translation when it contacts the translation system. Usually, a solid-phase binding site is provided in the spacer that links mRNA and PM, and the solid-phase binding site is fixed to the mRNA-PM conjugate via a “solid-phase binding site recognition site” that is bound to the solid phase. Fix to phase. The solid phase binding site is not particularly limited as long as it can bind the mRNA-PM conjugate to a desired solid phase. For example, a molecule (for example, a ligand, an antibody, etc.) that specifically binds to a specific polypeptide is used as such a solid-phase binding site. In this case, the solid-phase surface has a solid-phase binding site recognition site. The specific polypeptide that binds to the molecule is bound. Examples of combinations of solid phase binding sites / solid phase binding site recognition sites include, for example, biotin binding proteins / biotin such as avidin and streptavidin, maltose binding protein / maltose, G protein / guanine nucleotide, polyhistidine peptide / nickel or Metal ions such as cobalt, glutathione-S-transferase / glutathione, DNA binding protein / DNA, antibody / antigen molecule (epitope), calmodulin / calmodulin binding peptide, ATP binding protein / ATP, or estradiol receptor protein / estradiol, Examples include various receptor proteins / ligands thereof. Among these, combinations of solid phase binding site / solid phase binding site recognition site include biotin binding proteins such as avidin and streptavidin, maltose binding protein / maltose, polyhistidine peptide / metal ions such as nickel or cobalt, glutathione, etc. -S-transferase / glutathione, antibody / antigen molecule (epitope) and the like are preferable, and the combination of streptavidin / biotin is most preferable.
A known method can be used to bind the protein to the solid phase surface. Examples of such known methods include tannic acid, formalin, glutaraldehyde, pyruvic aldehyde, bis-diazotized benzidone, toluene-2,4-diisocyanate, amino group, carboxyl group, hydroxyl group or amino group. (P. M. Abdella, P. K. Smith, G. P. Royer, A New Creatable Reagent for Cross-Linking and Reversible Immobilization of Proteins, Biochem. BioR. Biochem. Bioy. 87, 734 (1979) etc.).
In addition, the said combination can also be used by reversing a solid-phase binding site and a solid-phase binding site recognition site. The above-described immobilization means is an immobilization method using two substances having an affinity for each other. If the solid phase is a plastic material such as a styrene bead or a styrene substrate, a known method can be used as necessary. Can also be used to covalently bond some of the spacers directly to their solid phase (see Qiagen, LiquiChip Applications Handbook et al.). In the present invention, the fixing means is not limited to the above method, and any fixing means known to those skilled in the art can be used.
3. Protein solid phase immobilization method and protein solid phase synthesis method
According to another aspect of the present invention, there is provided a method for solid phase immobilization or solid phase synthesis of a protein, wherein (c) the mRNA-PM conjugate is brought into contact with a translation system after the step (b). (For example, introducing a translation system into the ligated body or introducing the ligated body into the translation system) provides a solid phase immobilization or synthesis method of a protein, which includes a step of synthesizing a protein. In step (b), since the mRNA-PM conjugate is immobilized on the solid phase, when this conjugate is introduced into the translation system, it is synthesized using the association technique of the aforementioned in vitro virus method. The protein is immobilized on the solid phase via puromycin.
In the step (c), protein synthesis is performed by bringing the mRNA-PM conjugate into contact with a translation system. Examples of translation systems that can be used here include cell-free translation systems and living cells. As the cell-free translation system, a cell-free translation system composed of prokaryotic or eukaryotic extracts such as Escherichia coli, rabbit reticulocyte, wheat germ extract, etc. can be used (Lamfrom H, Grunberg-Manago M. Ambigites of translation of poly U in the rabbit reticulocyte system.Biochem Biophys Res Commun. 1967 27 (1): 1-6 etc.). As a living cell translation system, prokaryotic or eukaryotic organisms such as E. coli cells can be used. In the present invention, it is preferable to use a cell-free system because of easy handling.
Here, based on FIG. 1, protein immobilization using the mRNA-puromycin conjugate of the present invention will be briefly described. FIG. 1a is a diagram showing an immobilized mRNA-PM conjugate at the time of storage, FIG. 1b is a diagram showing a state in which a cell-free translation system is introduced and a protein is synthesized, and FIG. 1c is a state in which protein synthesis is completed. FIG. As shown in FIG. 1a, the immobilized mRNA-puromycin conjugate 1 is an mRNA 1a and puromycin 1b linked via a DNA spacer 1c and bound to the solid phase 2 via a solid phase binding site 1d. Has been. The solid phase 2 has a shape of a container in consideration of later introduction of a cell-free translation system. As shown in FIG. 1b, when a cell-free translation system 3 is introduced into the immobilized mRNA-puromycin conjugate 1, protein 4 corresponding to the nucleic acid sequence of the mRNA is obtained by a translation reaction using the mRNA and the cell-free translation system. Is synthesized. After the completion of translation, unnecessary components of the cell-free translation system 3 are removed, and an mRNA-puromycin-protein complex in which protein 4 is bound to puromycin 1b is formed in a state of being immobilized on solid phase 2. . In addition, the target substance which can be combined with the synthesized protein can be screened, for example, by bringing the target substance into contact with such an mRNA-puromycin-protein complex (conjugate).
4). mRNA chip and protein chip
According to another aspect of the present invention, the present invention relates to an mRNA chip (mRNA microarray) comprising a plurality of the above-described immobilized mRNA-puromycin conjugates. That is, the present invention provides an mRNA chip obtained by immobilizing the immobilized mRNA-puromycin conjugate of the present invention on a microarray substrate. This mRNA chip is obtained by immobilizing the above-described mRNA-PM conjugate on a plurality of substrates. The above “plurality” is not particularly limited as to the upper limit value, but refers to a practical number that can be fixed on a chip normally used as a microarray. By introducing this mRNA chip into the translation system, or by introducing the translation system into the mRNA chip, the above-described protein synthesis occurs on the chip, and so-called protein chips are produced in which each protein is bound to a solid phase. The The protein chip thus produced is also included in the present invention.
In the mRNA chip of the present invention, multiple mRNAs encoding proteins with known functions may be immobilized on the solid phase as mRNA-PM conjugates, or multiple mRNAs encoding proteins with unknown functions may be linked to mRNA-puromycin. You may fix to a solid phase as a body. For example, when mRNAs encoding proteins with known functions involved in diseases are immobilized on a plurality of chips, for example, a disease diagnosis mRNA chip, a protein interaction analysis mRNA chip or the like can be used. When used as a diagnostic chip, mRNA encoding a protein that binds to a diagnostic marker for a specific disease is fixed at a predetermined position on the plate. Then, a cell-free translation system is introduced into this plate immediately before diagnosis, and a protein that binds to a desired diagnostic marker is synthesized and fixed at a predetermined position on the plate. In this way, a protein chip can be produced immediately before diagnosis. Protein chips have problems in storage or handling. It can be said that one of the features of the present invention is that the chip is formed in the form of stable mRNA instead of unstable protein. Therefore, technologies other than solid phase immobilization of mRNA and protein synthesis / immobilization (for example, the material and size of the plate to be used, the type and arrangement of the protein to be used, the protein functional analysis method using the protein chip, etc.) The known protein chip technology can be used as it is (Kukar T, Eckenrod S, Gu Y, Lian W, Megginson M, She JX, Wu D. Protein microarrays to detect protein interaction in protein interaction Anal Biochem.2002; 306 (1): 50-4 etc.). In addition, a diagnostic kit including the mRNA beads or the mRNA chip and a cell-free translation system, or a protein interaction analysis kit is also within the scope of the present invention. By using such mRNA chip, it can be used for protein-protein interaction, DNA-protein interaction, ligand search, disease marker search, disease diagnosis, drug efficacy evaluation, pharmacokinetic evaluation and the like.
In addition, a conjugate comprising a structure in which a protein synthesized by bringing the immobilized mRNA-puromycin conjugate of the present invention into contact with a translation system is added to the conjugate (“immobilized mRNA-puromycin-protein linkage”). The term "body" is also included in the present invention. That is, the present invention relates to a protein (polypeptide) of the translation product of mRNA synthesized by subjecting the “immobilized mRNA-puromycin conjugate” of the present invention to a translation system, and puromycin or puromycin-like protein in the conjugate. The present invention relates to an immobilized mRNA-puromycin-protein conjugate linked through a compound. The conjugate can be suitably used, for example, in a “method for analyzing an interaction between a protein and a target molecule” described later. Further, as one embodiment of the above-mentioned linked body, as described above, a protein chip having a structure in which an immobilized mRNA-puromycin-protein coupled body is fixed to a microarray substrate can be exemplified.
Further, in a preferred embodiment of the present invention, the above-mentioned “immobilized mRNA-puromycin-protein conjugate” is subjected to a reverse transcription reaction system (contacted with the reverse transcription reaction system) to obtain a reverse transcription product of the mRNA. Complementary DNA (DNA that hybridizes to the mRNA) can be synthesized. For example, when a DNA spacer containing a sequence complementary to the 3 ′ end sequence of mRNA is used as the spacer of the present invention, the complementary sequence is expected to function as a primer in the reverse transcription reaction of the mRNA. . That is, by subjecting the mRNA-puromycin-protein conjugate to a reverse transcription reaction system, a DNA synthesis reaction using the primer as a synthesis starting point is started, and DNA having a sequence complementary to the mRNA is synthesized. A conjugate containing the DNA synthesized in this way (described as “DNA-puromycin-protein conjugate”) is also included in the present invention. That is, the present invention is an immobilization wherein the complementary DNA of the mRNA synthesized by subjecting the “immobilized mRNA-puromycin-protein conjugate” of the present invention to a reverse transcription reaction system is bound to the conjugate. DNA-puromycin-protein conjugates are provided.
The nucleic acid molecule linked to puromycin in the ligated body of the present invention is usually a double-stranded nucleic acid molecule of mRNA and a complementary DNA of the mRNA, but the mRNA in the nucleic acid molecule is a subsequent nuclease reaction or the like. May have been digested by. That is, the “immobilized DNA-puromycin-protein conjugate” comprising a structure in which a single-stranded DNA molecule (complementary to mRNA) is linked to puromycin is also an embodiment of the conjugate of the present invention. . Furthermore, in the above-mentioned ligated product of the present invention, the nucleic acid molecule linked to puromycin may be a double-stranded DNA consisting of DNA having complementarity with the DNA.
In the present invention, the “reverse transcription reaction system” refers to a reaction system that controls so-called “reverse transcription” in which DNA is synthesized using mRNA as a template, and the reaction system usually contains a reverse transcriptase. The reverse transcription reaction of the present invention can be appropriately performed by those skilled in the art. More specifically, it can be carried out by the method described in the examples below.
5). Methods for analyzing protein-molecule interactions
According to another aspect of the present invention, a method for analyzing an interaction between a protein and a molecule using the mRNA-PM conjugate described above is provided. This analysis method is
(A) a step of introducing one or more immobilized mRNA-puromycin conjugates of the present invention into a translation system and synthesizing a protein on a solid phase;
(B) contacting the protein synthesized in step (a) with one or more target substances; and
(C) including a step of measuring whether or not the protein and the target substance interact with each other.
This analysis method is used, for example, when (i) screening a substance that acts on a protein with a known sequence, or (ii) screening a protein with an unknown sequence to which a specific substance (for example, a ligand) binds. Can do. For example, in the case of (i), a plurality of conjugates of mRNA having a nucleic acid sequence encoding a protein with a known sequence (for example, orphan receptor protein) and puromycin are prepared (that is, a plurality of orphan receptor proteins). A plurality of mRNA-PM conjugates each having an mRNA corresponding to 1) are prepared, and this is put into a translation system. Then, a plurality of orphan receptor proteins are synthesized from the mRNA of each mRNA-PM conjugate. Each orphan receptor protein is immobilized by binding the C-terminus to puromycin of an mRNA-PM conjugate immobilized on a solid phase. If necessary, unnecessary components are washed and removed, and a target substance, a buffer, and the like are added thereto, and the target substance is bound to the orphan receptor protein to perform a binding experiment. In the case of (ii), for example, a plurality of mRNAs are obtained from a certain gene library, a conjugate of a plurality of mRNAs and puromycin is prepared and immobilized on a solid phase. Thereafter, the protein is synthesized in the same manner, and a binding test is performed by bringing the target substance into contact with the protein.
In the step (b), the protein synthesized in the step (a) is brought into contact with one or more target substances. The “target substance” used herein means a substance for examining whether or not it interacts with the protein synthesized in the present invention, and specifically includes proteins, nucleic acids, sugar chains, low molecular weight compounds, and the like. It is done.
There is no restriction | limiting in particular as protein, The partial peptide containing the binding active site may be sufficient as the full length of protein. Further, the protein may be a protein whose amino acid sequence and its function are known or unknown. These can also be used as target molecules, such as synthesized peptide chains, proteins purified from living organisms, or proteins that have been translated using a suitable translation system from a cDNA library or the like and purified. The synthesized peptide chain may be a glycoprotein having a sugar chain bound thereto. Of these, a purified protein having a known amino acid sequence or a protein translated and purified from a cDNA library or the like using an appropriate method can be preferably used.
The nucleic acid is not particularly limited, and DNA or RNA can also be used. Further, it may be a nucleic acid with a known base sequence or function or an unknown nucleic acid. Preferably, the nucleic acid having the ability to bind to a protein and the nucleotide sequence are known, or those that have been cleaved and isolated from a genomic library or the like using a restriction enzyme or the like can be used.
The sugar chain is not particularly limited, and its sugar sequence or function may be a known sugar chain or an unknown sugar chain. Preferably, a sugar chain that has already been separated and analyzed and whose sugar sequence or function is known is used.
The low molecular compound is not particularly limited, and a compound having an unknown function or a compound already known for its ability to bind to a protein can be used.
The “interaction” between these target substance and protein usually means at least one of covalent bond, hydrophobic bond, hydrogen bond, van der Waals bond and electrostatic force bond between protein and target molecule. The term indicates the effect of the forces acting between the molecules, but this term should be interpreted in the broadest sense and should not be construed as limiting in any way. The covalent bond includes a coordination bond and a dipole bond. In addition, electrostatic coupling includes electric repulsion in addition to electrostatic coupling. In addition, a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above action are also included in the interaction. Specific examples of interactions include binding and dissociation between antigen and antibody, binding and dissociation between protein receptor and ligand, binding and dissociation between adhesion molecule and partner molecule, binding and dissociation between enzyme and substrate, Binding and dissociation between nucleic acids and proteins that bind to them, binding and dissociation between proteins in information transmission systems, binding and dissociation between glycoproteins and proteins, or binding and dissociation between sugar chains and proteins Is mentioned.
The target substance used here can be used after being labeled with a labeling substance as necessary. If necessary, labeling can be performed by binding a labeling substance. Such a labeling substance is appropriately selected from fluorescent substances, radioactive labeling substances and the like. As the fluorescent substance, various fluorescent dyes having free functional groups (for example, a carboxyl group that can be converted into an active ester, a hydroxyl group that can be converted into a phosphoramidide, or an amino group) and that can be linked to a target substance can be used. . Suitable labeling substances include, for example, fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate, dansyl chloride or tetramethylrhodamine isothiocyanate; ³ H, ¹⁴ C, ¹²⁵ I or ¹³¹ And radioisotopes such as I. As these labeling substances, those suitable for measuring or analyzing a change in signal generated based on the interaction between the target substance and the immobilized protein are appropriately used. The labeling substance can be bound to the target substance based on a known method.
Next, according to this analysis method, in step (c), it is measured whether or not the protein and the target substance interact. Whether or not the protein and the target substance are interacting is measured by measuring and detecting a change in signal generated based on the interaction between the two molecules. Examples of such measurement techniques include surface plasmon resonance (Cullen, DC, et al., Biosensors, 3 (4), 211-225 (1987-88)), evanescent field molecular imaging (Funatsu). , T., et al., Nature, 374, 555-559 (1995)), fluorescent imaging analysis method, solid-phase enzyme immunoassay (ELISA): Crowther, JR, Methods in Molecular. Biology, 42 (1995)), fluorescence depolarization (Perran, J., et al., J. Phys. Rad., 1, 390-401 (1926)), and fluorescence correlation spectroscopy (FCS): Eigen. M., et al., Proc. Natl. Acad. Sci. 91 , 5740-5747 (1994)).
In the analysis method of the present invention, the protein and / or target substance in the protein-target substance conjugate determined to interact in step (c) is further identified as necessary. Protein identification can be performed by a normal amino acid sequencer, or by reverse transcription of DNA from mRNA bound to the protein and analysis of the base sequence of the obtained DNA. The target substance can be identified by NMR, IR, various mass spectrometry and the like. When analyzing the protein-protein interaction using the mRNA chip and protein chip of the present invention, the time-of-flight mass spectrometer (MALDI-TOF MS) is used in the same manner as the sample analysis on a normal protein chip. Can be used.
Moreover, the method of the present invention is complementary to the mRNA in the conjugate by subjecting the mRNA-puromycin-protein conjugate synthesized in the step (a) to the reverse transcription reaction system following the step (a). A DNA-containing conjugate (DNA-puromycin-protein conjugate) may be prepared and the subsequent steps described above may be performed. That is, in a preferred embodiment of the present invention, a method for analyzing an interaction between a protein and a molecule including the following steps is provided.
(A) contacting one or more of the immobilized mRNA-puromycin conjugate according to any of the present invention and a translation system to synthesize a protein on a solid phase;
(B) contacting the mRNA-puromycin-protein conjugate synthesized in step (a) with a reverse transcription reaction system to prepare a DNA-puromycin-protein conjugate;
(C) contacting the DNA-puromycin-protein conjugate prepared in step (b) with one or more target substances; and
(D) a step of measuring whether or not the protein in the conjugate and the target substance interact;

FIG. 1 is a schematic diagram showing a method for synthesizing and immobilizing a protein using the immobilized mRNA-puromycin conjugate of the present invention.
FIG. 2 is a diagram showing the two types of genes used in Examples 1 and 2.
FIG. 3 is a diagram showing a schematic diagram of a protein A B-domain or GFP mRNA obtained by covalently binding a puromachined spacer DNA.
4 is a photograph showing the results of SDS-PAGE in Example 1. FIG.
FIG. 5 is a photograph of the fluorescence of GFP translated on the beads in Example 2 observed under a microscope.

Explanation of symbols

1: immobilized mRNA-puromycin conjugate, 1a: mRNA,
1b: puromycin, 1c: DNA spacer,
1d: solid phase binding site, 2: solid phase, 3: cell-free translation system,
4: Protein.

  Hereinafter, the present invention will be described more specifically based on examples. The present invention is not limited to these examples.

０．２Ｍリン酸バッファー（ｐＨ７．０）１００μｌに、５００ｐｍｏｌ／μｌ Ｂｉｏｔｉｎ−ｌｏｏｐ［（５６ｍｅｒ）配列；５’−ＣＣＣＧＧ ＴＧＣＡＧ ＣＴＧＴＴ ＴＣＡＴＣ（Ｔ−Ｂ）ＣＧＧＡ ＡＡＣＡＧ ＣＴＧＣＡ ＣＣＣＣＣ ＣＧＣＣＧ ＣＣＣＣＣ ＣＧ（Ｔ）ＣＣＴ−３’（配列番号１、ＢＥＸ社より購入），（Ｔ）：Ａｍｉｎｏ−Ｍｏｄｉｆｉｅｒ Ｃ６ ｄＴ，（Ｔ−Ｂ）：Ｂｉｏｔｉｎ−ｄＴ（アンダーラインは制限酵素ＰｖｕＩＩのサイトを示す）］２０μｌ、１００ｍＭ架橋剤ＥＭＣＳ（３４４−０５０５１；６−Ｍａｌｅｉｍｉｄｏｈｅｘａｎｏｉｃ ａｃｉｄ Ｎ−ｈｙｄｒｏｘｙｓｕｃｃｉｎｉｄｅ ｅｓｔｅｒ、Ｄｏｊｉｎｄｏ社製）２０μｌ、を加え、良く攪拌した後、３７℃で３０分放置した後に、未反応のＥＭＣＳを取り除いた。沈殿を減圧下で乾燥させた後、０．２Ｍリン酸バッファー（ｐＨ７．０）１０μｌに溶かし、上記の還元したＰｕｒｏ−Ｆ−Ｓ（〜１０ｎｍｏｌ）を加えて４℃で一晩放置した。サンプルに最終で４ｍＭになるようにＴＣＥＰを加え室温で１５分放置した後、未反応のＰｕｒｏ−Ｆ−Ｓをエタノール沈殿で取り除き、未反応のＢｉｏｔｉｎ−ｌｏｏｐを取り除くために以下の条件でＨＰＬＣ精製を行った。
カラム；ｎａｃａｌａｉ ｔｅｓｑｕｅ ＣＳＯＭＯＳＩＬ ３７９１８−３１ １０ｘ２５０ｍｍ Ｃ１８−ＡＲ−３００（Ｗａｔｅｒｓ）
ＢｕｆｆｅｒＡ；０．１Ｍ ＴＥＡＡ、ＢｕｆｆｅｒＢ；８０％アセトニトリル（超純水で希釈したもの）
流速；０．５ｍｌ／ｍｉｎ（Ｂ％：１５−３５％ ３３ｍｉｎ）
ＨＰＬＣの分画は１８％アクリルアミドゲル（８Ｍ尿素、６２℃）で解析し、目的の分画を減圧下で乾燥させた後、ＤＥＰＣ処理水で溶かして、１０ｐｍｏｌ／μｌにした。
２．固相上での翻訳（ＰｒｏｔｅｉｎＡ Ｂ−ｄｏｍａｉｎ）
２−１ 実験に使用する遺伝子のｍＲＮＡを合成
５’側にＴ７、Ｃａｐ、オメガ配列、Ｋｏｚａｋ配列、３’末端に６ｘヒスチジンタグをもち、終止コドンを削ったＰｒｏｔｅｉｎＡ Ｂ−ｄｏｍａｉｎ（３７１ｂｐ；配列番号２）及び５’末端にＴ７、Ｃａｐ、Ｋｏｚａｋ配列をもち終止コドンを削ったＧＦＰ（Ｇｒｅｅｎ ｆｌｕｏｒｅｓｃｅｎｔ ｐｒｏｔｅｉｎ）（７１７ｂｐ；配列番号３、Ｉｔｏ，ｅｔ ａｌ．，Ｂｉｏｃｈｅｍ Ｂｉｏｐｈｙｓ Ｒｅｓ Ｃｏｍｍｕｎ．１９９９：２６４（２）：５５６−６０に記載の変異体）をクローニングし使用した。共に、スペーサーＤＮＡの一部と相補的であるタグ配列（５’側−ａｇｇａｃｇｇｇｇｇｇｃｇｇｇｇａａａ（配列番号４）、アンダーラインはスペーサー配列と相補的な部分）を含むように設計したプライマーでＰＣＲ反応を行うことで、３’末端にタグ配列をもつＤＮＡを得た。ＰＣＲ反応はＴａＫａＲａ ＥｘＴａｑ（ＴａｋａｒａＢｉｏ社）１ユニットを５０μｌのＰＣＲ反応液に加え、鋳型ＤＮＡは１ｆｍｏｌ加え、下記プライマーを用い、下記条件で行った。

［条件］熱変性９５℃ ２分の後、熱変性９５℃ ３０秒、
アニーリング６９℃ １５秒、伸長反応７２℃ ４５秒
のサイクルを３０回繰返し
得られたＤＮＡの構成を図２に示す。後に、ｉｎ ｖｉｔｒｏ転写（Ｐｒｏｍｅｇａ、Ｐ１３００）でｍＲＮＡを合成した。転写は、プロメガ社のキット付属のプロトコルに従いＤＮＡ１μｇ、２０μｌスケールで次のように行った。すなわち、３７℃で一時間放置した後、キット付属のＤＮａｓｅ（ＲＱ１ ＤＮａｓｅ）を１ユニット加え、さらに３７℃で１５分放置した。合成の際、プロメガ社のプロトコルに従い、ｍ７Ｇ Ｃａｐ Ａｎａｌｏｇ（Ｐｒｏｍｅｇａ，Ｐ１７１１）を加えた。５’キャップアナログがついたｍＲＮＡは、ＤＮａｓｅ、フェノール・クロロフォルム処理した後、ＤＳ Ｐｒｉｍｅｒ Ｒｅｍｏｖｅｒ（Ｅｄｇｅ Ｂｉｏｓｙｓｔｅｍｓ）で精製し定量した。
２−２ ｍＲＮＡとＰＭ付きスペーサーＤＮＡの結合
５’キャップ及び３’にタグ配列を持ったｍＲＮＡ ３００ｐｍｏｌに、ＰＭ付きスペーサーＤＮＡを３００ｐｍｏｌ、１０ｘＬｉｇａｔｉｏｎ Ｂｕｆｆｅｒ（ＴａＫａＲａ）６μｌ、ＤＭＳＯ ２．５μｌ、を加え５５μｌになるようＤＥＰＣ処理水を加えた。熱湯上で８５℃から３５℃へ２０分かけてアニーリングし、１５ｕｎｉｔ Ｔ４ Ｐｌｙｎｕｃｌｅｏｔｉｄｅ Ｋｉｎａｓｅ（ＴａＫａＲａ、２．５μｌ）、１００ｕｎｉｔ Ｔ４ ＲＮＡ Ｌｉｇａｓｅ（ＴａＫａＲａ、２．５μｌ）を加え、２５℃４５分反応させた。サンプルをＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ（Ｑｕｉａｇｅｎ，７４１０４）で処理した後、さらにＤＳ Ｐｒｉｍｅｒ Ｒｅｍｏｖｅｒで精製した。
図３に、ＰｒｏｔｅｉｎＡ Ｂ−ｄｏｍａｉｎまたはＧＦＰのｍＲＮＡへ、ＰＭ付きスペーサーＤＮＡを共有結合させたものの概略図を示す。図３中、Ｐはピューロマイシン、ＦはＦＩＴＣ、Ｂはビオチン、ＡＴＣＧｕはＤＮＡ、ＲＮＡシーケンスを示している。大文字で示した部分は制限酵素ＰｖｕＩＩサイト（四角枠で囲った部分）を含むＤＮＡ部分、小文字で示した部分はｍＲＮＡで、３’末端側のＤＮＡと相補鎖を形成している部分がＴａｇ配列に結合している。ＲＮＡの３’末端とＤＮＡの５’末端は“Ｔ４ ＲＮＡ Ｌｉｇａｓｅ”と示した部分でライゲートされている。
２−３ ＰＭ付きスペーサーＤＮＡ−ｍＲＮＡ複合体のビーズ上への結合
上記のように合成した、ＰＭ付きスペーサーＤＮＡとｍＲＮＡの複合体を、直径２．３μｍ±０．３μｍのアビジンビーズ（ＭＡＧＮＯＴＥＸ−ＳＡ、ＴａＫａＲａ、９０８８）へ、添付のプロトコルに基づき以下のようにして結合させた。
６０μｌのアビジンビーズを２００μｌの１ ｘ Ｂｉｎｄｉｎｇ Ｂｕｆｆｅｒ（添付されたもの）で２回、マグネットスタンドを用いてアビジンビーズを沈殿させ、上清を交換することで洗浄した。洗浄後、沈殿させたビーズへ、上記２−２で合成したＰＭ付きスペーサーＤＮＡとｍＲＮＡの複合体を４８ｐｍｏｌ加え、１ ｘ Ｂｉｎｄｉｎｇ Ｂｕｆｆｅｒ（添付されたもの）を合計で１２０μｌになるように加え、１０分間室温で静置した。その後、上記のように、２００μｌの１ ｘ Ｂｉｎｄｉｎｇ Ｂｕｆｆｅｒ（添付されたもの）でビーズを洗浄することで、ビーズに結合しなかったＰＭ付きスペーサーＤＮＡとｍＲＮＡの複合体を取り除いた。さらに、２０ ｘ Ｔｒａｎｓｌａｔｉｏｎ Ｍｉｘ（Ａｍｂｉｏｎ）１０μｌ、ＤＥＰＣ処理水１９０μｌを加え、同様にビーズを洗浄した。
２−４ 翻訳
上記のビーズをマグネットスタンド上で沈殿させ、無細胞翻訳系（Ｒｅｔｉｃ Ｌｙｓａｔｅ ＩＶＴ Ｋｉｔ，Ａｍｂｉｏｎ社，１２００）を３００μｌ分加え、３０℃１５分翻訳反応を行った。その後、ＭｇＣｌ_２、ＫＣｌをそれぞれ最終で６３ｍＭ、７５０ｍＭになるように加えて３７℃で１．５時間放置した。サンプルは、約１時間毎に軽く攪拌した。上記のようにビーズを沈殿させ、ＳＵＰＥＲａｓｅ・Ｉｎ（Ａｍｂｉｏｎ社、２６９４）２０ｕｎｉｔを含んだ１ ｘ Ｂｉｎｄｉｎｇ Ｂｕｆｆｅｒ（添付されたもの）２００μｌで２回ビーズを洗浄した。
２−５ 逆転写
洗浄後、上記と同様にして沈殿させたビーズに対して、ＴａＫａＲａ ＢＩＯＭＥＤＩＣＡＬＳ社添付のプロトコルに従い、８０μｌのスケールで逆転写酵素Ｍ−ＭＬＶ（ＴａＫａＲａ、２６４０Ａ）を用いて４２℃１０分間、逆転写反応を行った。その後に上記と同様にして、１ ｘ Ｂｉｎｄｉｎｇ Ｂｕｆｆｅｒ（添付されたもの）２００μｌでビーズを洗浄した。
２−６ ビーズからＤＮＡ−タンパク質を回収
沈殿させたビーズに対して、添付のプロトコルに従い、４０μｌのスケールで２４ｕｎｉｔの制限酵素ＰｖｕＩＩ（ＴａＫａＲａ）で３７℃１時間放置することでビーズ上のＤＮＡ−タンパク質をビーズから切り離す処理を行った。ここでは特に、ビーズとＤＮＡ−タンパク質の非特異的な吸着を避けるために、ＢＳＡを最終０．１ｍｇ／ｍｌになるように加えた。その後、上記と同様にしてビーズを沈殿させ、上清を新しいサンプルチューブに移した。逆転写の際、テンプレートとなったｍＲＮＡがＤＮＡ−タンパク質のＤＮＡの部分と相補鎖を作ったままなので、その上清にＲｉｂｏｎｕｃｌｅａｓｅ Ｈ（Ｐｒｏｍｅｇａ，Ｍ４２８１）を２ｕｎｉｔ加え、３７℃、２０分反応させることで逆転写後に残っているｍＲＮＡ部分を分解した。
２−７ Ｈｉｓタグ精製
Ｑｕｉａｇｅｎ社のプロトコルに従い、サンプルを等量のＬｙｓｉｓバッファー（ＮａＨ_２ＰＯ_４ ５０ｍＭ，ＮａＣｌ ３００ｍＭ，ｉｍｉｄａｚｏｌｅ １０ｍＭ，Ｔｗｅｅｎ２０ ０．０５％，ｐＨ８．０）に希釈し、２０μｌのＮｉ−ＮＴＡビーズ（Ｎｉ−ＮＴＡ ＭａｇｎｅｔｉｃＡｇａｒｏｓｅＢｅａｄｓ、ＱＩＡＧＥＮ社、３６１１１）を加え、室温で４０分攪拌しながら放置した。上記と同様にしてマグネットスタンドでＮｉ−ＮＴＡビーズを沈殿させ、１００μｌのＷａｓｈバッファー（ＮａＨ_２ＰＯ_４ ５０ｍＭ，ＮａＣｌ ３００ｍＭ，ｉｍｉｄａｚｏｌｅ ２０ｍＭ，Ｔｗｅｅｎ２０ ０．０５％，ｐＨ８．０）で軽く洗浄した。ビーズを沈殿させた後、Ｅｌｕｔｅバッファー（ＮａＨ_２ＰＯ_４ ５０ｍＭ，ＮａＣｌ ３００ｍＭ，ｉｍｉｄａｚｏｌｅ ２５０ｍＭ，Ｔｗｅｅｎ２０ ０．０５％，ｐＨ８．０）を１５μｌ加え１分間室温で放置し、ＤＮＡ−タンパク質を溶出させた。同様にしてビーズを沈殿させ、ＤＮＡ−タンパク質が溶出した上清をとりわけ、６Ｍ尿素を含んだ５％ＳＤＳ−ＰＡＧＥで解析した。ゲルのバンドは、Ｍｏｌｅｃｕｌａｒ ｉｍａｇｅｒ ＦＸ（Ｂｉｏ ＲＡＤ ｃｏ．）でＦＩＴＣの蛍光を可視化することで定量した。得られた結果を図４に示す。
３．固相上でのタンパク質合成（ＰｒｏｔｅｉｎＡ Ｂ−ｄｏｍａｉｎ）の結果
図４ａは、ＰＭ付きスペーサーＤＮＡとｍＲＮＡの複合体を無細胞翻訳系に加え、溶液中で（通常どおりに）ｍＲＮＡ−タンパク質の複合体を作成した結果であり、ＦＩＴＣの蛍光を可視化したものである。レーン１は翻訳前、２は翻訳後である。翻訳反応後現れた“ＲＮＡ ｖｉｒｕｓ”と示した位置のバンドは、その分子量から判断して、スペーサーＤＮＡのピューロマイシンを介して、ｍＲＮＡとそのｍＲＮＡがコードするタンパク質が共有結合された複合体であると考えられる。“ｇｅｎｏｍｅ”と記した位置のバンドは、タンパク質が結合していない、スペーサーＤＮＡ、ｍＲＮＡの複合体である。
ビーズ上で翻訳、逆転写反応を行い、制限酵素でビーズから切り離し回収したものも同様に、２本のバンドが観察され（レーン１）、高分子量のバンド（矢じり）が目的とするＤＮＡ−タンパク質の共有結合体、低分子量の太いバンドはタンパク質がつかなかったものであると考えられる（図４ｂ）。このことは、タンパク質のＣ末端側に導入した６ ｘ Ｈｉｓ−ｔａｇで精製した結果、高分子量のバンドのみが溶出されたことから確認される（レーン２、３）。また、この結果は、ここで合成・固定化されたＤＮＡ−タンパク質複合体のタンパク質部分は、通常液相で合成されるタンパク質同様、完全長が合成されたことを示すものである。
これらの結果から、固相上に、ＰＭ付きスペーサーＤＮＡを介してｍＲＮＡを固定し、そこに無細胞翻訳系を加える方法で、そのｍＲＮＡがコードしているタンパク質を翻訳直後に自動的に固相上に固定化できることが示された。
なお、ＳＤＳ−ＰＡＧＥにおけるＦＩＴＣの蛍光で可視化したバンドを基に回収されたＤＮＡ−タンパク質複合体の量を計測した結果、固相上で合成され制限酵素で切り離し回収した時点でのＤＮＡ−タンパク質（図４ｂ矢じり）は、加えたｍＲＮＡの０．４％、さらにＨｉｓ−ｔａｇ精製し抽出されたもの（図４ｂ“ＤＮＡ ｖｉｒｕｓ”）は、加えたｍＲＮＡの０．１％だった。
〔実施列２〕固相上で合成されたタンパク質の機能を検出（ＧＦＰ）
１．スペーサーがついたｍＲＮＡをビーズ上へ固定
実施例１の２−３と同様にＰＭ付きスペーサーＤＮＡとｍＲＮＡの複合体をアビジンビーズに固定した。ＧＦＰの蛍光を観察するために、スペーサーＤＮＡは上記２で用いたものとは違い、Ｐｕｒｏ−Ｓ［配列；５’−（Ｓ）−ＴＣ（Ｆ）−（Ｓｐｅｃ１８）−（Ｓｐｅｃ１８）−（Ｓｐｅｃ１８）−（Ｓｐｅｃ１８）−ＣＣ−（Ｐｕｒｏ）−３’、（Ｓ）：５’−Ｔｈｉｏｌ−Ｍｏｄｉｆｉｅｒ Ｃ６、（Ｐｕｒｏ）：Ｐｕｒｏｍｙｃｉｎ ＣＰＧ、ＢＥＸ社より購入］で同様に合成したものを使用した。アビジンビーズはＢａｎｇｓ社の直径４６０ｎｍのものを使用し、以下のようにしてビーズ上に固定化した。
１０μｌ分のビーズを１００μｌの０．５ ｘ Ｂｉｎｄｉｎｇ ｂｕｆｆｅｒ（５０ｍＭ Ｔｒｉｓ ＨＣｌ ｐＨ８．０，０．０５％ Ｔｗｅｅｎ２０，５００ｍＭ ＮａＣｌ）で２度洗浄した。洗浄は、ビーズを含んだ溶液を１５，０００ｒｐｍ ５分間４℃で遠心分離し、上清を液交換することで行った。洗浄したビーズの沈殿に、実施例１の２−２と同様の方法で合成した、８ｐｍｏｌのＰＭ付きスペーサーＤＮＡとＧＦＰのｍＲＮＡの複合体を加え、１ ｘ Ｂｉｎｄｉｎｇ Ｂｕｆｆｅｒで４０μｌになるように希釈し、室温で１５分静置してビーズに結合させた。上記のようにして１００μｌの０．５ ｘ Ｂｉｎｄｉｎｇ ｂｕｆｆｅｒで１度洗浄し、ビーズに結合しなかったｍＲＮＡを取り除いた。さらに、２０ ｘ ＴｒａｎｓＬａｔｉｏｎＭｉｘ（Ａｍｂｉｏｎ社）１０μｌ、ＤＥＰＣ処理水１９０μｌを加え、同様にビーズを洗浄した。
２．翻訳
遠心分離で沈殿させたビーズへ無細胞翻訳系（Ｒｅｔｉｃ Ｌｙｓａｔｅ ＩＶＴ Ｋｉｔ，Ａｍｂｉｏｎ社，１２００）を２０μｌ分加え、３０℃３０分反応させた後、ＭｇＣｌ_２、ＫＣｌをそれぞれ最終で６３ｍＭ、７５０ｍＭになるように加え、２５℃で２時間放置した。サンプルは、約１時間毎に軽く攪拌した。１５，０００ｒｐｍ ６分遠心分離によりビーズを沈殿させ、上記と同様にして遠心分離により、リン酸ＮａＣｌバッファー（５０ｍＭリン酸ナトリウムｐＨ７．０、１００ｍＭ ＮａＣｌ）で２回ビーズを洗浄した。沈殿させたビーズを２０μｌリン酸ＮａＣｌバッファーに懸濁し、氷上で保存した。
３．顕微鏡下での観察
上記のビーズを含む懸濁液を５０ｍＭリン酸バッファーｐＨ７．０でさらに１／５に希釈し、活性酸素除去のための酵素系（２５ｍＭ ｇｌｕｃｏｓｅ，２．５μＭ ｇｌｕｃｏｓｅ ｏｘｉｄａｓｅ，１０ｎＭ ｃａｔａｌａｓｅ，１０ｍＭ ｄｉｔｉｏｔｈｒｅｉｔｏｌ）存在下で顕微観察を行った。顕微鏡はＮｉｃｏｎ、ＴＥ２０００を用い、４７３ｎｍ ０．３５ｍＷの対物エバネッセント照明で励起し、ＧＦＰの蛍光を冷却ＣＣＤカメラＯＲＣＡ−ＥＲ（浜松フォトニクス）で１．０４秒の露光で撮影した。ネガティブなコントロールとして、実施例１の３−１及び３−２と同じ方法で並列してビーズにスペーサーＤＮＡのみを同じ濃度で結合させ、同じ条件で観察を行った。観察結果を図５に示す。図５は、ビーズの上で翻訳させたＧＦＰの蛍光を顕微鏡下で観察した写真である。ビーズ（直径４６０ｎｍ）の明視野像に、４７３ｎｍのレーザーで励起したときの蛍光像を重ね合わせた。図５ａは、ＰＭ付きスペーサーＤＮＡとＧＦＰのｍＲＮＡの複合体８ｐｍｏｌを１０μｌ分のアビジンビーズに結合させ翻訳反応を行ったもの、図５ｂは、ＰＭ付きＤＮＡスペーサーのみ８ｐｍｏｌを１０μｌ分のアビジンビーズに結合させ、同じ条件で翻訳反応を行った結果の顕微観察像を示す。蛍光像は緑の擬似カラーで確認した。
４．結果
実施例１の結果をさらに、確かなものにするために、同じ方法でＧＦＰをビーズ上で翻訳し、翻訳されたビーズ上のＧＦＰの蛍光をエバネッセント顕微鏡下で直視することを試みた。その結果、ＰＭ付きスペーサーＤＮＡのみをビーズに結合させ観察を行ったときに比べて優位に、ＰＭ付きスペーサーＤＮＡにＧＦＰのｍＲＮＡをつないだ後ビーズに固定し翻訳させたビーズ上に、ＧＦＰの蛍光を観察することができた。したがって、目的とするタンパク質が固相で合成され固定されたことが再度確認された。
産業上の利用の可能性
以上説明したように、本発明によれば、ｍＲＮＡ及びタンパク質の固相固定化方法、固定化ｍＲＮＡ−ピューロマイシン連結体、この連結体を含むｍＲＮＡビーズ又はｍＲＮＡチップ、このｍＲＮＡチップから作製されるプロテインチップなどを提供することができる。このようなｍＲＮＡチップは、保存が容易であるから、既存のプロテインチップと比較して極めて取り扱いが容易であるという利点がある。
また本発明は、上記固定化ｍＲＮＡ−ピューロマイシン連結体を翻訳系へ供して合成される該ｍＲＮＡの翻訳産物のタンパク質が、該ピューロマイシンと結合した構造の「固定化ｍＲＮＡ−ピューロマイシン−タンパク質連結体」を提供する。該連結体は、タンパク質と相互作用し得る分子の解析あるいはスクリーニングに利用することができる。
さらに本発明は、上記「固定化ｍＲＮＡ−ピューロマイシン−タンパク質連結体」を、逆転写反応系へ供することにより、該ｍＲＮＡの逆転写産物の相補的ＤＮＡが連結した構造の「固定化ＤＮＡ−ピューロマイシン−タンパク質連結体」を提供する。通常、ＲＮＡに比べてＤＮＡはより安定であることから、タンパク質相互作用解析において、該連結体を被検分子と接触させる際に、該連結体は非常に有用である。
また、本発明のｍＲＮＡチップは、各種疾病の診断マーカーを認識するタンパク質の合成に用いられるｍＲＮＡを固定することによって、各種疾病の診断に利用することができる。さらに、本発明のタンパク質の固相固定化又は合成方法は、タンパク質と分子との相互作用の解析にも好適に利用できる。1. Synthesis of Spacer BioLoop-Puro (hereinafter abbreviated as “spacer DNA with PM”) Puro-FS [sequence; 5 ′-(S) -TC (F)-(Spec18)-(Spec18)-(Spec18)-( Spec18) -CC- (Puro) -3 ′, purchased from BEX] 10 nmol was dissolved in 100 μl of 50 mM phosphate buffer (pH 7.0), 1 μl of 100 mM TCEP (final 1 mM) was added, and left at room temperature for 6 hours. Purio-FS Thiol was reduced. TCEP (Tris (2-carboxyethyl) phosphine hydrochloride) was removed using NAP5 (Amersham, 17-0853-02) equilibrated with 50 mM phosphate buffer (pH 7.0) immediately before the crosslinking reaction. In the sequence of Puro-FS, (S) is 5'-Thiol-Modifier C6, (F) is Fluorescein-dT, (Puro) is Puromycin CPG, and Spacer 18 is a spacer (18-) made by Glen Research Search. O-Dimethyltriethylhexyleneglycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramide) has the following chemical structure.

In 0.2M phosphate buffer (pH7.0) 100μl, 500pmol / μl Biotin-loop [(56mer) sequence; 5'-CCCGG TG CAG CTG TT TCATC (T-B) CGGA AA CAG CTG CA CCCCC CGCCG CCCCC CG ( T) CCT-3 ′ (SEQ ID NO: 1, purchased from BEX), (T): Amino-Modifier C6 dT, (TB): Biotin-dT (underline indicates the site of restriction enzyme PvuII)] 20 μl , 20 μl of 100 mM cross-linking agent EMCS (344-05051; 6-Maleimidohexanoic acid N-hydroxysuccinide ester, manufactured by Dojindo), and after stirring well at 37 ° C. for 30 minutes, unreacted E Removal of the CS. The precipitate was dried under reduced pressure, dissolved in 10 μl of 0.2 M phosphate buffer (pH 7.0), and the reduced Puro-FS (10 nmol) was added and left at 4 ° C. overnight. TCEP was added to the final sample to 4 mM and left at room temperature for 15 minutes. Unreacted Puro-FS was removed by ethanol precipitation, and HPLC purification was performed under the following conditions to remove unreacted Biotin-loop. Went.
Column; nacalai tesque CSOMOSIL 37918-31 10 × 250 mm C18-AR-300 (Waters)
Buffer A; 0.1M TEAA, Buffer B; 80% acetonitrile (diluted with ultrapure water)
Flow rate: 0.5 ml / min (B%: 15-35% 33 min)
The HPLC fraction was analyzed on an 18% acrylamide gel (8M urea, 62 ° C.), and the desired fraction was dried under reduced pressure and then dissolved in DEPC-treated water to 10 pmol / μl.
2. Translation on solid phase (Protein A B-domain)
2-1 Synthesis of mRNA of gene used in experiment Protein A B-domain (371 bp; SEQ ID NO: 371) having T7, Cap, omega sequence, Kozak sequence at 5 'side, 6x histidine tag at 3' end and deletion of stop codon 2) and GFP (Green Fluorescent Protein) (717 bp; SEQ ID NO: 3, Ito, et al., Biochem Biophys Res Commun. 1999: 264 (2) with T7, Cap and Kozak sequences at the 5 ′ end and deletion of the stop codon. : 556-60 mutant) was cloned and used. Both PCR reactions should be performed with primers designed to include a tag sequence that is complementary to a part of the spacer DNA (5 ′ side-aggacgggggggggggggaa (SEQ ID NO: 4), underlined is a portion complementary to the spacer sequence). Thus, DNA having a tag sequence at the 3 ′ end was obtained. The PCR reaction was performed under the following conditions using 1 unit of TaKaRa ExTaq (TakaraBio) added to 50 μl of the PCR reaction solution, 1 fmol of template DNA, and the following primers.

[Conditions] Thermal denaturation at 95 ° C for 2 minutes, thermal denaturation at 95 ° C for 30 seconds,
Annealing 69 ° C 15 seconds, elongation reaction 72 ° C 45 seconds
The structure of DNA obtained by repeating this cycle 30 times is shown in FIG. Later, mRNA was synthesized by in vitro transcription (Promega, P1300). Transcription was performed as follows on a 1 μg DNA and 20 μl scale according to the protocol attached to the kit of Promega. That is, after standing at 37 ° C. for 1 hour, 1 unit of DNase (RQ1 DNase) attached to the kit was added, and the mixture was further left at 37 ° C. for 15 minutes. During the synthesis, m7G Cap Analog (Promega, P1711) was added according to the Promega protocol. MRNA with a 5 ′ cap analog was treated with DNase and phenol / chloroform, and then purified and quantified with DS Primer Remover (Edge Biosystems).
2-2 Binding of mRNA and spacer DNA with PM Add 300 μmol of PM spacer DNA with 5 ′ cap and 3 ′ tag sequence, add 6 μl of 10 × Ligation Buffer (TaKaRa), 2.5 μl of DMSO, and add 55 μl to 55 μl. DEPC-treated water was added. The mixture was annealed from 85 ° C. to 35 ° C. over hot water over 20 minutes, and 15 unit T4 Polynucleotide Kinase (TaKaRa, 2.5 μl) and 100 unit T4 RNA Ligase (TaKaRa, 2.5 μl) were added and reacted at 25 ° C. for 45 minutes. The sample was treated with RNeasy Mini Kit (Qiagen, 74104) and further purified with DS Primer Remover.
FIG. 3 shows a schematic diagram of ProteinA B-domain or GFP mRNA covalently bound with spacer DNA with PM. In FIG. 3, P represents puromycin, F represents FITC, B represents biotin, and ATCGu represents DNA and RNA sequences. The part shown in capital letters is the DNA part containing the restriction enzyme PvuII site (the part enclosed in a square frame), the part shown in small letters is mRNA, and the part that forms a complementary strand with the DNA on the 3 ′ end side is the Tag sequence. Is bound to. The 3 ′ end of RNA and the 5 ′ end of DNA are ligated at a portion indicated as “T4 RNA Ligase”.
2-3 Binding of PM spacer spacer DNA-mRNA complex onto beads The PM spacer spacer DNA-mRNA complex synthesized as described above was avidin bead (MAGNOTEX-SA having a diameter of 2.3 μm ± 0.3 μm. , TaKaRa, 9088) based on the attached protocol as follows.
60 μl of avidin beads were washed with 200 μl of 1 × Binding Buffer (attached) twice by precipitating the avidin beads using a magnetic stand and exchanging the supernatant. After washing, 48 pmol of the complex of spacer DNA with PM and mRNA synthesized in 2-2 above was added to the precipitated beads, and 1 × Binding Buffer (attached) was added to a total of 120 μl. Let stand at room temperature for minutes. Thereafter, as described above, the bead was washed with 200 μl of 1 × Binding Buffer (attached) to remove the PM spacer spacer DNA / mRNA complex that did not bind to the beads. Further, 10 μl of 20 × Translation Mix (Ambion) and 190 μl of DEPC-treated water were added, and the beads were washed in the same manner.
2-4 Translation The above beads were precipitated on a magnet stand, a cell-free translation system (Retic Lysate IVT Kit, Ambion, 1200) was added for 300 μl, and a translation reaction was performed at 30 ° C. for 15 minutes. Thereafter, MgCl ₂ and KCl were added to 63 mM and 750 mM, respectively, and left at 37 ° C. for 1.5 hours. Samples were gently agitated approximately every hour. The beads were precipitated as described above, and the beads were washed twice with 200 μl of 1 × Binding Buffer (supplied) containing SUPERase In (Ambion, 2694) 20 units.
2-5 Reverse transcription After washing and precipitating in the same manner as described above, the reverse transcriptase M-MLV (TaKaRa, 2640A) was used at 42 ° C. at a scale of 80 μl according to the protocol attached to TaKaRa BIOMEDICALS. Reverse transcription reaction was performed for a minute. Thereafter, the beads were washed with 200 μl of 1 × Binding Buffer (attached) in the same manner as described above.
2-6 Recovery of DNA-protein from beads By following the attached protocol, the DNA-protein on the beads is allowed to stand at 37 ° C. for 1 hour with 24 units of restriction enzyme PvuII (TaKaRa) in a scale of 40 μl according to the attached protocol. Was removed from the beads. Here, in particular, BSA was added to a final concentration of 0.1 mg / ml to avoid non-specific adsorption of beads and DNA-protein. Thereafter, beads were precipitated as described above, and the supernatant was transferred to a new sample tube. During reverse transcription, the template mRNA remains complementary to the DNA portion of the DNA-protein. Add 2 units of Ribonuclease H (Promega, M4281) to the supernatant and react at 37 ° C for 20 minutes. The mRNA portion remaining after reverse transcription was degraded.
2-7 His tag purification According to the protocol of Qiagen, the sample was diluted in an equal volume of Lysis buffer (NaH ₂ PO ₄ 50 mM, NaCl 300 mM, imidazole 10 mM, Tween 20 0.05%, pH 8.0), and 20 μl of Ni- NTA beads (Ni-NTA Magnetic Agarose Beads, QIAGEN, 36111) were added, and the mixture was allowed to stand at room temperature with stirring for 40 minutes. Ni-NTA beads were precipitated with a magnetic stand in the same manner as described above, and lightly washed with 100 μl of Wash buffer (NaH ₂ PO ₄ 50 mM, NaCl 300 mM, imidazole 20 mM, Tween 20 0.05%, pH 8.0). After the beads were precipitated, 15 μl of Elute buffer (NaH ₂ PO ₄ 50 mM, NaCl 300 mM, imidazole 250 mM, Tween 20 0.05%, pH 8.0) was added and left at room temperature for 1 minute to elute the DNA-protein. In the same manner, beads were precipitated, and the supernatant from which the DNA-protein was eluted was analyzed by, inter alia, 5% SDS-PAGE containing 6M urea. Gel bands were quantified by visualizing FITC fluorescence with a Molecular imager FX (Bio RAD co.). The obtained results are shown in FIG.
3. Results of protein synthesis on solid phase (Protein A B-domain) FIG. 4a shows that a complex of spacer DNA with PM and mRNA is added to a cell-free translation system, and a complex of mRNA-protein in solution (as usual). This is a result of visualization of FITC fluorescence. Lane 1 is before translation and 2 is after translation. The band at the position indicated as “RNA virus” that appears after the translation reaction is a complex in which mRNA and the protein encoded by the mRNA are covalently bound via puromycin, a spacer DNA, as judged from its molecular weight. it is conceivable that. The band at the position marked “genome” is a complex of spacer DNA and mRNA to which no protein is bound.
Similarly, two bands are observed (lane 1) after the translation and reverse transcription reaction on the beads, and the fragments separated from the beads with a restriction enzyme (lane 1). It is considered that the low-molecular-weight thick band of this covalent bond was not attached to the protein (FIG. 4b). This is confirmed from the fact that only the high molecular weight band was eluted as a result of purification with 6 × His-tag introduced on the C-terminal side of the protein (lanes 2 and 3). This result also shows that the full length of the protein part of the DNA-protein complex synthesized and immobilized here was synthesized in the same manner as a protein synthesized in a normal liquid phase.
From these results, by immobilizing mRNA on a solid phase via spacer DNA with PM and adding a cell-free translation system there, the protein encoded by the mRNA is automatically immobilized on the solid phase immediately after translation. It was shown that it can be immobilized on top.
The amount of the DNA-protein complex recovered based on the band visualized by FITC fluorescence in SDS-PAGE was measured. As a result, the DNA-protein (synthesized on the solid phase, separated by the restriction enzyme and recovered) In FIG. 4b (arrowhead), 0.4% of the added mRNA was further purified and extracted with His-tag (FIG. 4b “DNA virus”) was 0.1% of the added mRNA.
[Example 2] Detecting the function of a protein synthesized on a solid phase (GFP)
1. Immobilization of mRNA with spacers on beads As in 2-3 of Example 1, a complex of spacer DNA with PM and mRNA was immobilized on avidin beads. In order to observe the fluorescence of GFP, the spacer DNA was different from that used in 2 above, and Puro-S [sequence; 5 ′-(S) -TC (F)-(Spec18)-(Spec18)-(Spec18] )-(Spec18) -CC- (Puro) -3 ′, (S): 5′-Thiol-Modifier C6, (Puro): Puromycin CPG, purchased from BEX, Inc.] was used. Avidin beads having a diameter of 460 nm from Bangs were used and immobilized on the beads as follows.
Ten μl of beads were washed twice with 100 μl of 0.5 × Binding buffer (50 mM Tris HCl pH 8.0, 0.05% Tween 20, 500 mM NaCl). Washing was performed by centrifuging the solution containing the beads at 15,000 rpm for 5 minutes at 4 ° C., and exchanging the supernatant. To the precipitate of the washed beads, a complex of 8 pmol PM-attached spacer DNA with PM and GFP mRNA synthesized in the same manner as in Example 2-2 was added, and diluted to 40 μl with 1 × Binding Buffer. The mixture was allowed to stand for 15 minutes at room temperature to bind to the beads. As described above, the plate was washed once with 100 μl of 0.5 × Binding buffer to remove mRNA that did not bind to the beads. Further, 10 μl of 20 × TransLationMix (Ambion) and 190 μl of DEPC-treated water were added, and the beads were washed in the same manner.
2. Translation After adding 20 μl of a cell-free translation system (Retic Lysate IVT Kit, Ambion, 1200) to the beads precipitated by centrifugation and reacting at 30 ° C. for 30 minutes, MgCl ₂ and KCl were finally added to 63 mM and 750 mM, respectively. And left at 25 ° C. for 2 hours. Samples were gently agitated approximately every hour. The beads were precipitated by centrifugation at 15,000 rpm for 6 minutes, and the beads were washed twice with a phosphate buffer (50 mM sodium phosphate pH 7.0, 100 mM NaCl) by centrifugation as described above. The precipitated beads were suspended in 20 μl NaCl phosphate buffer and stored on ice.
3. Observation under a microscope The suspension containing the above beads was further diluted to 1/5 with 50 mM phosphate buffer pH 7.0, and an enzyme system for removing active oxygen (25 mM glucose, 2.5 μM glucose oxidase, 10 nM catalase). , 10 mM dithiothreitol) was observed. The microscope was a Nico, TE2000, excited with 473 nm 0.35 mW objective evanescent illumination, and GFP fluorescence was taken with a cooled CCD camera ORCA-ER (Hamamatsu Photonics) at an exposure of 1.04 seconds. As a negative control, only the spacer DNA was bound to the beads at the same concentration in parallel in the same manner as in Examples 3-1 and 3-2, and observation was performed under the same conditions. The observation results are shown in FIG. FIG. 5 is a photograph of the fluorescence of GFP translated on the beads observed under a microscope. A fluorescent image when excited with a 473 nm laser was superimposed on a bright field image of beads (diameter 460 nm). Fig. 5a shows a translation reaction with 8pmol of a spacer DNA with PM and GFP mRNA bound to 10µl of avidin beads, and Fig. 5b shows that only 8pmol of PM spacer with PM is bound to 10µl of avidin beads. A microscopic image of the result of translation reaction under the same conditions is shown. The fluorescent image was confirmed with a green pseudo color.
4). Results In order to further confirm the results of Example 1, we tried to translate GFP on the beads in the same way and to look directly at the fluorescence of GFP on the translated beads under an evanescent microscope. As a result, compared with the case where only the spacer DNA with PM was bound to the beads and observed, GFP mRNA was fixed on the beads after translation of the GFP mRNA to the spacer DNA with PM and translated. Was able to be observed. Therefore, it was confirmed again that the target protein was synthesized and immobilized in the solid phase.
As described above, according to the present invention, the solid phase immobilization method of mRNA and protein, the immobilized mRNA-puromycin conjugate, the mRNA bead or mRNA chip containing this conjugate, A protein chip or the like produced from an mRNA chip can be provided. Since such mRNA chip is easy to store, it has the advantage that it is extremely easy to handle compared to existing protein chips.
The present invention also provides a structure in which the protein of the translation product of the mRNA synthesized by subjecting the immobilized mRNA-puromycin conjugate to the translation system is bound to the puromycin “immobilized mRNA-puromycin-protein linkage”. "Body" is provided. The conjugate can be used for analysis or screening of molecules that can interact with proteins.
Furthermore, the present invention provides the above-mentioned “immobilized mRNA-puromycin-protein conjugate” to a reverse transcription reaction system, whereby “immobilized DNA-puro having a structure in which complementary DNAs of the reverse transcription product of the mRNA are linked. A “mycin-protein conjugate” is provided. Since DNA is usually more stable than RNA, the conjugate is very useful when contacting the conjugate with a test molecule in protein interaction analysis.
In addition, the mRNA chip of the present invention can be used for diagnosis of various diseases by immobilizing mRNA used for the synthesis of proteins that recognize diagnostic markers for various diseases. Furthermore, the solid phase immobilization or synthesis method of the protein of the present invention can be suitably used for analyzing the interaction between the protein and the molecule.

[Sequence Listing]

Claims (17)

An immobilized mRNA-puromycin conjugate comprising a conjugate of mRNA and puromycin or puromycin-like compound immobilized on a solid phase. The immobilized mRNA-puromycin conjugate according to claim 1, wherein the mRNA-puromycin conjugate is obtained by linking puromycin or a puromycin-like compound to the 3 'end of mRNA via a spacer. The immobilized mRNA-puromycin conjugate according to claim 1 or 2, wherein the mRNA-puromycin conjugate is bound to a solid phase via a solid phase binding site provided in the spacer. The immobilized mRNA-puromycin conjugate according to any one of claims 1 to 3, wherein the spacer comprises a polynucleotide, polyethylene, polyethylene glycol, polystyrene, peptide nucleic acid, or a combination thereof as a main skeleton. The solid phase is selected from styrene beads, glass beads, agarose beads, sepharose beads, magnetic beads, glass substrates, silicon substrates, plastic substrates, metal substrates, glass containers, plastic containers, and membranes. The immobilized mRNA-puromycin conjugate according to any one of the above. A protein of a translation product of the mRNA synthesized by subjecting the immobilized mRNA-puromycin conjugate according to any one of claims 1 to 5 to a translation system, via puromycin or a puromycin-like compound in the conjugate. An immobilized mRNA-puromycin-protein conjugate, An immobilized DNA-puro obtained by subjecting the immobilized mRNA-puromycin-protein conjugate according to claim 6 to a reverse transcription reaction system and a complementary DNA of the mRNA bound to the conjugate. Mycin-protein conjugate. An mRNA chip comprising the immobilized mRNA-puromycin conjugate according to any one of claims 1 to 5 immobilized on a microarray substrate. A protein chip produced using the mRNA chip according to claim 8. An mRNA bead in which a conjugate of mRNA and puromycin or a puromycin-like compound is immobilized on the bead. A kit for protein interaction analysis comprising the mRNA chip according to claim 8 or the mRNA bead according to claim 10 and a cell-free translation system. (A) linking mRNA and puromycin via a spacer provided with a solid phase binding site to prepare an mRNA-puromycin conjugate, and
(B) A method of immobilizing mRNA on a solid phase, comprising the step of immobilizing the mRNA-puromycin conjugate on a solid phase by binding the solid phase binding site of the spacer to the solid phase. (A) a step of preparing an mRNA-puromycin conjugate by linking mRNA and puromycin via a spacer provided with a solid phase binding site;
(B) fixing the mRNA-puromycin conjugate to the solid phase by binding the solid phase binding site of the spacer to the solid phase; and (c) the mRNA-puromycin conjugate and the translation system. A method for immobilizing a protein on a solid phase, comprising a step of synthesizing the protein by contacting the protein. (A) a step of preparing an mRNA-puromycin conjugate by linking mRNA and puromycin via a spacer provided with a solid phase binding site;
(B) fixing the mRNA-puromycin conjugate to the solid phase by binding the solid phase binding site of the spacer to the solid phase; and (c) the mRNA-puromycin conjugate and the translation system. A method of synthesizing a protein in a solid phase, comprising a step of synthesizing the protein by contacting. A method for analyzing the interaction between proteins and molecules,
(A) contacting one or more of the immobilized mRNA-puromycin conjugate according to any one of claims 1 to 5 with a translation system to synthesize a protein on a solid phase;
(B) contacting the protein synthesized in step (a) with one or more target substances; and (c) measuring whether or not the protein and the target substance interact;
Including the analysis method. A method for analyzing the interaction between proteins and molecules,
(A) contacting one or more of the immobilized mRNA-puromycin conjugate according to any one of claims 1 to 5 with a translation system to synthesize a protein on a solid phase;
(B) contacting the mRNA-puromycin-protein conjugate synthesized in step (a) with a reverse transcription reaction system to prepare a DNA-puromycin-protein conjugate;
(C) contacting the DNA-puromycin-protein conjugate prepared in step (b) with one or more target substances; and (d) the protein in the conjugate interacts with the target substance. Measuring whether or not;
Including the analysis method. Furthermore, the analysis method of Claim 15 or 16 including the process of identifying the protein and / or target substance which were judged to be interacting.

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