Simple and convenient G-quadruplex-based fluorescent assay of biotin-streptavidin interaction via terminal protection of small molecule-linked DNA

Abstract

Small molecule–protein interaction plays a critical role in a majority of chemical genetics, clinical diagnostics, and drug development. Herein, by using biotin–streptavidin interaction assay as a model, we describe a strategy for simple and accurately determining streptavidin (SA) activity by means of a coupled terminal protection strategy and G-quadruplex-based platform. A G-rich ssDNA [G3(T4G3)3] with a tail (T12) at the 3′ end (5′-GGGTTTTGGGTTTTGGGTTTTGGGTTTTTTTTTTTT-3′) has been adopted. Biotin was labeled at the 3′ end of the ssDNA. In the absence of streptavidin (SA), the biotinylated ssDNA is digested in the 3′ to 5′ direction by Exo I to form mononucleotides. The formation of the G-quadruplex is prohibited due to the lack of the G-rich ssDNA, and this results in weak fluorescence of N-methyl mesoporphyrin IX (NMM). Conversely, in the presence of SA, the specific binding of SA to the biotinylated ssDNA protects the ssDNA from digestion. Afterward, the G-rich region of the ssDNA folds into a stable G-quadruplex in the presence of potassium ions, thus strongly enhancing the fluorescence of NMM. This assay has a wide linear range and low detection limit. The performance of SA detection in 10 % serum sample has also been investigated. The method not only provides a universal platform for monitoring small molecule–protein interaction but also shows great potential in chemistry, biology, and medicine.