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 dic. 2020
Accepted
13 feb. 2021
First published
16 feb. 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

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements