構造の切り替えは不要:小分子検出のための一般化可能なエクソヌクレアゼ媒介アプタマーベースのアッセイ

Juan Canoura¹, Zongwen Wang^1,2, Haixiang Yu¹

¹Department of Chemistry and Biochemistry , Florida International University , 11200 Southwest Eighth Street , Miami , Florida 33199 , United States.

Journal of the American Chemical Society

|July 17, 2018

まとめ

この研究は,アプタマーとエクソヌクレアスを用いた小分子検出の新しい方法を導入しています. 標的結合はアプタマーを消化から保護し,シーケンスエンジニアリングなしで敏感でラベルのない検出を可能にします.

科学分野:

生物化学
分子生物学
分析化学

背景:

DNAに結合する小分子は,その外核酵素の消化に対する感受性を変化させることができる.
アプタマーとは短いDNAやRNAの配列で分子認識や検出に用いられる.

研究の目的:

アプタマーとエクソヌクレアスを用いた微小分子検出のための敏感でラベルフリーな方法を開発する.
この新しい検査の広範な適用性とマルチプレキシング能力を実証する.

主な方法:

標的結合時にエクソヌクレアゼIIIの消化に抵抗するプリフォールドアプタマーを使用する.
選択的な製品生成のために二重エクソヌクレアゼシステム (エクソヌクレアゼIIIとエクソヌクレアゼI) を使用する.
SYBRゴールドのような光染料を用いて残った二重鎖DNAを定量化します

主要な成果:

標的結合はエクソヌクレアゼIIIの消化を阻害し,検出可能な二重鎖製品を残します.
非標的結合アプタマーが分解され,標的結合アプタマーが無傷のままである.
デヒドロアイソアンドロステロン-3硫酸,コカイン,ATPの検出に敏感であることが示され,同時に多重検出も含まれています.

結論:

この二重エクソヌクレアスのアプタマー測定は,小分子検出のための一般的で敏感でラベルフリーなプラットフォームを提供します.
この方法は,様々なアプタマーとターゲットに適応し,生物学的マトリックスに適しています.
幅広い分析アプリケーションのための多様な信号報告戦略との統合の可能性.

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