Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, P. R. China.
Anal Methods. 2021 Mar 28;13(12):1489-1494. doi: 10.1039/d0ay02315a. Epub 2021 Mar 9.
In this work, homo-FRET (Förster resonance energy transfer between the same kind of fluorophores) takes place in a hetero-FRET (FRET between two different fluorophores) system and can effectively improve the energy transfer efficiency. Herein, a novel ratiometric fluorescence method was developed for the detection of nuclease activity. Exonuclease III (Exo III), an enzyme which has a high exodeoxyribonuclease activity for double-stranded DNA (dsDNA) in the 3' to 5' direction, was chosen as a proof of concept of this strategy. In a linear dsDNA template, the occurrence of homo-FRET in two Cy3 donors enables the highly efficient transfer of energy to the Cy5 acceptor. The ratio of fluorescence intensity between Cy3 and Cy5 (F/F) increases in an Exo III concentration-dependent manner, which built the foundation of Exo III quantification. This method exhibits a linear range from 0.25 to 8 U mL with a detection limit of 0.17 U mL. Importantly, this platform also shows the potential for screening Exo III inhibitors and detecting Exo III activity in complex samples.
在这项工作中，同型Förster 共振能量转移（同种荧光团之间的Förster 共振能量转移）发生在异型Förster 共振能量转移（两种不同荧光团之间的 FRET）系统中，可以有效提高能量转移效率。本文开发了一种用于检测核酸酶活性的新型比率荧光法。外切核酸酶 III（Exo III）是一种具有双链 DNA（dsDNA）3'到 5'方向的高外切脱氧核糖核酸酶活性的酶，被选为该策略的概念验证。在线性 dsDNA 模板中，两个 Cy3 供体中的同型 FRET 的发生使得能量能够高效地转移到 Cy5 受体上。Cy3 和 Cy5 之间的荧光强度比（F/F）与 Exo III 浓度呈依赖性增加，这为 Exo III 的定量奠定了基础。该方法在 0.25 至 8 U mL 的线性范围内具有检测限为 0.17 U mL。重要的是，该平台还显示了筛选 Exo III 抑制剂和检测复杂样品中 Exo III 活性的潜力。
