Issue 12, 2021

Homo-FRET enhanced ratiometric fluorescence strategy for exonuclease III activity detection

Abstract

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 (FD/FA) 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−1 with a detection limit of 0.17 U mL−1. Importantly, this platform also shows the potential for screening Exo III inhibitors and detecting Exo III activity in complex samples.

Graphical abstract: Homo-FRET enhanced ratiometric fluorescence strategy for exonuclease III activity detection

Supplementary files

Article information

Article type
Paper
Submitted
21 Dec. 2020
Accepted
13 Febr. 2021
First published
16 Febr. 2021

Anal. Methods, 2021,13, 1489-1494

Homo-FRET enhanced ratiometric fluorescence strategy for exonuclease III activity detection

X. Zhang, Y. Bai, Y. Jiang, N. Wang, F. Yang, L. Zhan and C. Huang, Anal. Methods, 2021, 13, 1489 DOI: 10.1039/D0AY02315A

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