Detection of biotin-streptavidin interactions via terminal protection of small molecule linked DNA and the formation of fluorescent DNA-templated silver nanoclusters

It is known that the binding of certain proteins to small molecules in ssDNA/small-molecule chimeras protects the conjugated ssDNA from degradation by exonuclease I (Exo I). This has resulted in numerous methods to specifically detect the interaction between small molecules and proteins. We are presenting here an approach that utilizes the terminal protection strategy in combination with the formation of ssDNA-templated silver nanoclusters (AgNCs), thereby providing a fluorometric tool for the detection of such interactions. A C-rich ssDNA (type 5′-CCCCACCCCT-3′) was labelled with biotin at the 3′ end. In the absence of streptavidin (SA), the biotinylated ssDNA is hydrolyzed in the 3′ to 5′ direction by Exo I to form mononucleotides. The formation of the AgNCs is prevented due to the lack of the DNA scaffold, and this results in weak fluorescence. Conversely, in the presence of SA, the specific binding of SA to the biotinylated ssDNA protects the ssDNA from digestion. As a result, fluorescent AgNCs are being formed. Fluorescence is measured at excitation/emission wavelengths of 625/705 nm. The calibration plot for SA is linear in the 6 to 600 nM concentration range, with a 2.6 nM detection limit. The assay is simple, sensitive and affordable. Conceivably, the method may also be used to detect the binding of other small molecules to proteins.

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Graphical abstract A fluorescent sensing platform for small molecule-protein interaction assay has been developed based on terminal protection strategy and ssDNA-templated silver nanoclusters (AgNCs).