変異した地域またはエナチオ選択性を持つRNA分裂DNA酵素

Phillip Ordoukhanian¹, Gerald F Joyce

¹Departments of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

Journal of the American Chemical Society

|October 17, 2002

まとめ

DNA酵素は,非自然な核酸の特定のフォスフォディエステル結合を裂くために進化しました. これらの新しいDNA触媒は,高い地域選択性とエナチオ選択性を示し,合成生物学における能力を拡大しています.

科学分野:

バイオケミストリーバイオケミストリー
分子生物学は分子生物学である.
合成生物学合成生物学とは

背景:

DNA酵素 (DNAzymes) は,触媒的に活性なDNA分子である.
In vitro進化は,新しい核酸触媒を生成するための強力なツールです.
フォスフォディエステル結合の特定の分裂は,分子生物学と治療において極めて重要です.

研究の目的:

特定のフォスフォディエステル結合を分裂できるDNA酵素を進化させる.
これらの新しいDNA酵素の触媒効率,地域選択性,およびエナチオ選択性を調査する.
非天然の基質のための触媒を作り出すための in vitro 進化の有用性を実証する.

主な方法:

触媒活性のあるDNA配列を選択するために,in vitro進化を用いた.
運動パラメータ (kcat,Km) と触媒効率 (kcat/Km) を決定するために測定を行った.
レジオ・セレクティブとエナチオセレクティブは,自然および非自然な核酸基板の割れ目を比較することによって評価されました.

主要な成果:

2つのDNA酵素が成功裏に進化した:一つは2',5'-フォスフォディエステル分裂,もう一つはL-リボヌクレオチドの3',5'-フォスフォディエステル結合分裂.
2',5'-phosphodiester-cleaving酵素は,高い触媒効率 (kcat/Km ~ 10^8 M^-1 min^-1) と地域選択性 (>6000倍) を示した.
L-リボヌクレオチド分裂酵素は,有意なエナチオセレクティブ性 (>40倍) を示し,二価金属カチオンを必要とした.

結論:

In vitro進化は,非自然な基質のための非常に特定のDNA酵素を生成することができます.
これらのDNA酵素は,非自然なヌクレオチドアナログに適用できる,顕著な地域およびエナチオセレクティブ性を示しています.
進化したDNA酵素は,合成生物学やそれ以上の応用のための有望なツールを表しています.

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