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Label-free fluorescence detection of protein–ligand interactions based on binding-induced enzymatic cleavage protection

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Abstract

Due to significant roles in cellular functions and cancer treatments, the development of efficient methods to monitor protein–ligand interactions is essential. Herein, we developed a highly selective fluorescence strategy for protein activity detection using a ligand-modified DNA and nucleic acid dye SYBR Green I (SGI). In order to verify our approach, streptavidin (SA) and biotin were selected as model molecules to be investigated. In the presence of targeted protein SA, the biotin-modified hpDNA can bind with SA and protect the hpDNA from enzymatic degradation. Then, the intact hpDNA can excite the fluorescence signal of free SGI. However, the unprotected hpDNA would undergo hydrolysis reactions to produce short fragments in the presence of Exo III and fail to excite the fluorescence of the SGI dye. So, the fluorescence change of SGI can be used to identify SA activity and SA–biotin interaction. This high-specific and label-free fluorescence method can detect protein in buffers as well as biological samples. Our proposed method provided good selectivity and high sensitivity with a detection limit of 0.001 μg mL−1 for SA detection. We believe that this strategy presents a promising platform for high-throughput protein–ligand interaction screening. Moreover, this method can also be applied to monitor other proteins by replacing ligands.

Graphical abstract: Label-free fluorescence detection of protein–ligand interactions based on binding-induced enzymatic cleavage protection

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Supplementary files

Article information


Submitted
02 Jul 2020
Accepted
29 Sep 2020
First published
03 Oct 2020

New J. Chem., 2020, Advance Article
Article type
Paper

Label-free fluorescence detection of protein–ligand interactions based on binding-induced enzymatic cleavage protection

L. Duan, H. Yao, Y. Xie and K. Pan, New J. Chem., 2020, Advance Article , DOI: 10.1039/D0NJ03316B

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