JP4463554B2 - 短い二本鎖RNAのinvitro合成方法 - Google Patents
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Description
Caplen,N.ら、2001、PNAS(98)9742−9747 Elbashir S.ら、2001、Nature(411)494−498 Milligan F.ら、1987、Nucleic Acid Res.(15)8783−8798
本発明は、a)鋳型で伸長されたセンスオリゴリボヌクレオチド産物が形成されるような反応混合物中でセンスの標的特異的オリゴヌクレオチド鋳型および鎖伸長酵素を組み合わせること;b)鋳型で伸長されたアンチセンスオリゴリボヌクレオチド産物が形成されるような反応混合物中でアンチセンスの標的特異的オリゴヌクレオチド鋳型および鎖伸長酵素を組み合わせること;ならびにc)段階a)で得られるセンスオリゴリボヌクレオチド産物を段階b)で得られる相補的アンチセンスオリゴリボヌクレオチド産物とハイブリダイズさせること、の段階を含んで成る短い二本鎖の標的特異的なRNAのin vitro合成方法を提供する。
本発明は短い二本鎖標的特異的RNAの合成の分野に関し、そして鎖伸長酵素を使用するオリゴヌクレオチド鋳型のin vitro転写に基づく。
1)標的遺伝子のコーディング配列内に配置されかつ以下の配列5’−xx(n12−30)yy−3’[式中xはプロモーターから転写されたヌクレオチドを指し、yはプロモーター配列から転写されたヌクレオチドに相補的なヌクレオチドを指し、そしてn12−30は12ないし30ヌクレオチドのいずれかのオリゴヌクレオチドを指す]を有する標的特異的配列を探す
2)場合によっては2個の付加的なヌクレオチドを伴い伸長された段階1)で配置された標的特異的配列の相補物オリゴヌクレオチド配列を伴い鋳型鎖の5’端で伸長された本発明の二本鎖RNAポリメラーゼプロモーター配列を含んで成るセンスオリゴヌクレオチド鋳型を設計する。
3)場合によっては2個の付加的なヌクレオチドを伴い伸長された段階1)で配置された標的特異的配列の逆オリゴヌクレオチド配列を伴い鋳型鎖の5’端で伸長された本発明の二本鎖RNAポリメラーゼプロモーター配列を含んで成るアンチセンスオリゴヌクレオチド鋳型を設計する
を含んで成る。
1)標的遺伝子のコーディング配列内に配置されかつ以下の配列5’−gg(n12−30)cc−3’を有する標的特異的配列を探す;
2)場合によっては2個の付加的なヌクレオチドを伴い伸長された以下の配列
5’TAATACGACTCACTATAGG
3’ATTATGCTGAGTGATATcc(n相補物)12−30gg[式中(n相補物)12−30は段階1)で配置された標的特異的配列の相補物オリゴヌクレオチド配列を指す]を有するセンスオリゴヌクレオチド鋳型を設計する;ならびに
3)場合によっては2個の付加的なヌクレオチドを伴い伸長された以下の配列
5’TAATACGACTCACTATAGG
3’ATTATGCTGAGTGATATcc(n逆)12−30gg[式中(n逆)12−30は段階1)で配置された標的特異的配列の逆オリゴヌクレオチド配列を指す]を有するアンチセンスオリゴヌクレオチド鋳型を設計する
を含むことができる。
1)標的遺伝子のコーディング配列内に配置されかつ以下の配列5’−xx(n15−30)yy−3’[式中xはプロモーターから転写されたヌクレオチドを指し、yはプロモーター配列から転写されたヌクレオチドに相補的なヌクレオチドを指し、そしてn15−30は15ないし30ヌクレオチドのいずれかのオリゴヌクレオチドを指す]を有する標的特異的配列を探す;
2)2個の付加的なヌクレオチド、好ましくは2個のアデニン残基を伴い伸長される段階1)で配置された標的特異的配列の相補物オリゴヌクレオチド配列を伴い鋳型鎖の5’端で伸長された本発明の二本鎖RNAポリメラーゼプロモーター配列を含んで成るセンスオリゴヌクレオチドsiRNA鋳型を設計する;
3)2個の付加的なヌクレオチド、好ましくは2個のアデニン残基を伴い伸長された段階1)で配置された標的特異的配列の逆オリゴヌクレオチド配列を伴い鋳型鎖の5’端で伸長された本発明の二本鎖RNAポリメラーゼプロモーター配列を含んで成るアンチセンスオリゴヌクレオチドsiRNA鋳型を設計する
を含むことができる。
1)標的遺伝子のコーディング配列内に配置されかつ以下の配列5’−gg(n15−30)cc−3’を有する標的特異的配列を探す;
2)以下の配列
5’TAATACGACTCACTATAGG
3’ATTATGCTGAGTGATATcc(n相補物)15−30ggaa
[式中(n相補物)15−30は段階1)で配置された標的特異的配列の相補物オリゴヌクレオチド配列を指す]を有するセンスオリゴヌクレオチドsiRNA鋳型を設計する;ならびに
3)以下の配列
5’TAATACGACTCACTATAGG
3’ATTATGCTGAGTGATATcc(n逆)15−30ggaa
[式中(n逆)15−30は段階1)で配置された標的特異的配列の逆オリゴヌクレオチド配列を指す]を有するアンチセンスオリゴヌクレオチドsiRNA鋳型を設計する
を含むことができる。
i)標的特異的センスおよびアンチセンスオリゴヌクレオチド鋳型の設計方法
ii)短い二本鎖RNAのin vitro合成方法での使用のための本発明の鎖伸長酵素
iii)センスおよびアンチセンスオリゴリボヌクレオチド産物を得るための精製手段。
iv)センスおよびアンチセンスオリゴリボヌクレオチド産物が本発明の鎖伸長酵素を使用して標的特異的センスおよびアンチセンス鋳型から形成されるような反応混合物のための試薬
を提供する。
材料および方法
プラスミド構築物
ルシフェラーゼ+はプラスミドpGL3−control(プロメガ(Promega))から発現させた。EGFPは哺乳動物発現ベクターpcDNA5/FRT(インヴィトロジェン(Invitrogen))のHindIIIおよびNotI部位に指向性に(directionally)連結されたpEGFP(クロンテック(Clonetech))からのEGFP遺伝子を含有するEGFP/pcDNA5−FRTから発現させた。
siRNAのin vitro転写およびハイブリダイゼーション
オリゴ鋳型鎖は2分間沸騰させかつ2〜3時間にわたって室温にゆっくり冷却することにより10mMトリス−HCl pH9.0、100mM NaCl、1mM EDTA中でセンスT7 プロモーター配列(5’TAATACGACTCACTATAGG)にハイブリダイズさせた。転写は製造元の説明書に従ってMEGAshortscriptTMT7キット(アンビオン(Ambion))を使用して実施した。siRNA鎖はG−25スピンカラムで精製し、重相ロックゲル(Heavy Phase−Lock Gel)(エッペンドルフ(Eppendorf))を使用してフェノール:クロロホルム:イソアミルアルコール(25:24:1)抽出し、そして−80℃で一夜エタノール沈殿した。相補的siRNA鎖を、2分間煮沸しかつ2〜3時間にわたって室温にゆっくり冷却することにより1mMトリス−HCl pH8.0、1mM EDTA pH8.0中でハイブリダイズさせた。ハイブリダイゼーションは非変性20%ポリアクリルアミドTBEゲル上でds−およびss−siRNAを泳動することにより評価した。
細胞系およびトランスフェクション
HeLa細胞は1.8mM l−グルタミン、9%FBSおよび45U/lペニシリン/ストレプトマイシンで補充した高ブドウ糖およびl−グルタミンを含むDMEM(インヴィトロジェン(Invitrogen))中で成長させた。細胞をElbashirら(2001)に記述されたものに類似の様式でトランスフェクトした。トランスフェクション24時間前に細胞をトリプシン処理しそして抗生物質を欠く成長培地で3×105細胞/mlに希釈した。0.5mlの細胞を24ウェルプレートの各ウェルに接種した。細胞は別の方法で示される場合を除き製造元の説明書に従ってリポフェクタミン[Lipofectamine]TM2000(LF2000;インヴィトロジェン(Invitrogen))を使用して1μgのGL3−controlもしくはEGFP/pcDNA5−FRTレポーター構築物および50pmolの一本鎖もしくは25pmolの二本鎖siRNAでトランスフェクトした。具体的には、われわれは抗生物質を欠く血清を含まない培地48μl中でウェルあたり2μlのLF2000を使用した。希釈されたLF2000を50μlの総容量に同一培地で希釈されたレポーターおよび/もしくはsiRNAと混合する前に1分間室温で前インキュベートした。その後、複合体を細胞に添加する前に20分間室温でインキュベートした。EGFPおよびGL3レポーター遺伝子アッセイを24時間後に実施した。JNK2α1 siRNA実験およびGL3 siRNA用量応答実験に6ウェルプレートを使用した。細胞数を4倍および試薬量を5倍増大させた。JNK2α1 siRNA実験には細胞をRNA単離のため収集し、また、タンパク質抽出はトランスフェクションおよそ48時間後であった。
レポーター遺伝子アッセイ
EGFPでトランスフェクトした細胞のFACSアッセイはFACScan(ベクトン・ディッキンソン(Beckton−Dickinson))を使用して実施した。細胞をトリプシン処理しそしてFACS固定溶液(PBS+1%ホルムアルデヒド)中への再懸濁前にPBSで洗浄した。トランスフェクション効率を、水でトランスフェクトしたサンプルをEGFP/pcDNA5−FRTでトランスフェクトしたものと比較することにより推定し、そして典型的には75〜90%であった。転写されたもしくは合成のsiRNAにより誘導されるRNAiの程度は、コトランスフェクトしたsiRNAを伴うもしくは伴わないサンプル中の平均GFP蛍光の変化から推定した。
ノーザンおよびウェスタンブロッティング
全RNAは製造元の説明書に従ってRNイージーミニキット(RNeasy Mini Kit)(キアゲン(Qiagen))を使用しておよそ106HeLa細胞のサンプルから調製した。サンプルを製造元の説明書に従ってプレキャストMOPSラチチュード(Latitude)RNAアガロースゲル(バイオウイタッカー モレキュラー アプリケーションズ(BioWhittaker Molecular Applications))上で泳動しそしてハイボンド(Hybond)−XLナイロンメンブレン(AP バイオテック(AP Biotech))に転写した。DNAプローブは製造元の説明書に従ってレディプライム(Rediprime)IIシステム(AP バイオテック(AP Biotech))を使用して作成した。ハイブリダイゼーションは製造元の説明書に従ってラピッド−ハイブ(Rapid−Hyb)溶液(AP バイオテック(AP Biotech))中で実施した。
結果
RNAiは2個の3’でオーバーハングするヌクレオチドを除き二本鎖であるsiRNAを使用して以前に立証されている(Elbashirら 2001;Caplenら 2001)。複数の細胞標的に対する多様なsiRNAを比較的迅速かつ安価に創製するために、われわれはin vitro転写技術を使用して分子を生成するためのスキームを設計した。
雄性Balb/Cマウス(およそ25g)(標準的住居、食餌/水への自由到達)は生理的食塩水2.3ml、もしくは800UのRNアーゼ阻害剤と一緒の短縮されたT7プロモーターのin vitro転写方法により調製されたマウスインスリン受容体に対し向けられたsiRNA(NCBI受託番号NM_010568;塩基2536−2556)40マイクログラムを含有する生理的食塩水のいずれかの尾静脈注入を受領した。
Claims (8)
- a)鋳型で伸長されたセンスオリゴリボヌクレオチド産物が形成されるような反応混合物中で、標的特異的センスオリゴヌクレオチド鋳型およびT7 RNAポリメラーゼを組み合わせること;
b)鋳型で伸長されたアンチセンスオリゴリボヌクレオチド産物が形成されるような反応混合物中で、標的特異的アンチセンスオリゴヌクレオチド鋳型およびT7 RNAポリメラーゼを組み合わせること;ならびに
c)段階a)で得られるセンスオリゴリボヌクレオチド産物を段階b)で得られる相補的アンチセンスオリゴリボヌクレオチド産物とハイブリダイズさせることであって、
段階a)およびb)のオリゴヌクレオチド鋳型が、鋳型鎖の5'端で標的特異的鋳型配列を伴い伸長された以下の図1
に示されるところの短縮されたT7 RNAポリメラーゼプロモーター配列よりなるRNAポリメラーゼプロモーター配列を含んで成り、ここで前記標的特異的鋳型配列が5'端に2個のグアノシン(g)ヌクレオチドおよび3'端に2個のシトシン(c)ヌクレオチドを含んで成る、
ことを特徴とする、
の段階を含んで成る、30ヌクレオチド長未満の標的特異的な短い二本鎖RNAの合成方法。 - 段階a)およびb)のオリゴヌクレオチド鋳型が、請求項1記載の図1に示されるところの二本鎖の短縮されたT7 RNAポリメラーゼプロモーター配列を含んで成る部分的に二本鎖のDNAオリゴ鋳型であることをさらに特徴とする、請求項1記載の方法。
- a)鋳型で伸長されたセンスオリゴリボヌクレオチド産物が形成されるような反応混合物中で、センスsiRNA鋳型をT7 RNAポリメラーゼと組み合わせること;b)鋳型で伸長されたアンチセンスオリゴリボヌクレオチド産物が形成されるような反応混合物中で、アンチセンスsiRNA鋳型をT7 RNAポリメラーゼと組み合わせること;ならびに
c)段階a)で得られるセンスオリゴリボヌクレオチド産物を段階b)で得られるアンチセンスオリゴリボヌクレオチド産物とハイブリダイズさせること;
それにより、段階a)およびb)のsiRNA鋳型が、鋳型鎖の5'端で請求項1で定義されるところの標的特異的鋳型配列および2もしくは3個の付加的ヌクレオチドを伴い伸長された請求項1記載の図1に示されるところの短縮されたT7 RNAポリメラーゼプロモーター配列よりなる二本鎖RNAポリメラーゼプロモーター配列を含んで成る、
の段階を含んで成る、12〜30ヌクレオチドの低分子干渉RNA(siRNA)の合成方法。 - 短い二本鎖RNAがセンス配列5'−gg(n15-30)cc−3'[ここでgは(請求項1記載の図1に示されるところの)短縮されたT7 RNAポリメラーゼプロモーターから転写されたヌクレオチドグアノシンを指し、cは(請求項1記載の図1に示されるところの)短縮されたT7 RNAポリメラーゼプロモーター配列から転写されたヌクレオチドに相補的なヌクレオチドシトシンを指し、そしてn15-30は15ないし30ヌクレオチドのいずれかのオリゴヌクレオチドを指す]を有することを特徴とする標的特異的な短い二本鎖RNAを含んで成るRNAの、インビトロでの細胞中への導入を含んで成る、細胞中での標的遺伝子の発現の阻害方法。
- 標的特異的な短い二本鎖RNAが2もしくは3個の付加的ヌクレオチドを伴い3'端で伸長される、請求項4記載の方法。
- 標的遺伝子が、細胞遺伝子、内在性遺伝子、トランスジーンもしくは病原体からの遺伝子である、請求項4ないし5のいずれか1つ記載の方法。
- 請求項1ないし3のいずれか1つ記載の方法での使用のための反応混合物であって、
鋳型が、前記鋳型鎖の5'端で標的特異的鋳型配列を伴い伸長された請求項1記載の図1に示されるところの短縮されたT7 RNAポリメラーゼプロモーター配列よりなるRNAポリメラーゼプロモーター配列を含んで成り、ここで前記標的特異的鋳型配列は5'端に2個のグアノシン(g)ヌクレオチドおよび3'端に2個のシトシン(c)ヌクレオチドを含んで成ることを特徴とする、標的特異的センスオリゴヌクレオチド鋳型、および、
鋳型が、前記鋳型鎖の5'端で標的特異的鋳型配列を伴い伸長された請求項1記載の図1に示されるところの短縮されたT7 RNAポリメラーゼプロモーター配列よりなるRNAポリメラーゼプロモーター配列を含んで成り、ここで前記標的特異的鋳型配列は5'端に2個のグアノシン(g)ヌクレオチドおよび3'端に2個のシトシン(c)ヌクレオチドを含んで成ることを特徴とする、標的特異的アンチセンスオリゴヌクレオチド鋳型を含んでなる、
上記反応混合物。 - 標的特異的センスおよびアンチセンスオリゴヌクレオチド鋳型を設計するための方法であって、以下の段階;
1)標的遺伝子のコーディング配列内に配置されかつ以下の配列5'−xx(n12-30)yy−3'[式中xはプロモーターから転写されたヌクレオチドを指し、yはプロモーター配列から転写されたヌクレオチドに相補的なヌクレオチドを指し、そしてn12-30は12ないし30ヌクレオチドのいずれかのオリゴヌクレオチドを指す]を有する標的特異的配列を探す、
2)場合によっては2個の付加的なヌクレオチドを伴い伸長された、段階1)で配置された標的特異的配列の相補物オリゴヌクレオチド配列を伴い鋳型鎖の5'端で伸長された、本発明の二本鎖RNAポリメラーゼプロモーター配列を含んで成るセンスオリゴヌクレオチド鋳型を設計する、
3)場合によっては2個の付加的なヌクレオチドを伴い伸長された、段階1)で配置された標的特異的配列の逆オリゴヌクレオチド配列を伴い鋳型鎖の5'端で伸長された、本発明の二本鎖RNAポリメラーゼプロモーター配列を含んで成るアンチセンスオリゴヌクレオチド鋳型を設計する、
を含んで成る上記方法。
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WO2003040294A2 (en) | 2003-05-15 |
DE60207601D1 (de) | 2005-12-29 |
EP1444347B1 (en) | 2005-11-23 |
WO2003040294A3 (en) | 2003-12-24 |
JP2005508177A (ja) | 2005-03-31 |
US20040259097A1 (en) | 2004-12-23 |
AU2002338926B2 (en) | 2007-05-24 |
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