Endonucleolytic Inhibition Assay of DNA/Fok I Transducer as a Sensitive Platform for Homogeneous Fluorescence Detection of Small Molecule-Protein Interactions

This paper reported a novel homogeneous fluorescence assay strategy for probing small molecule protein interactions based on endonucleolytic inhibition of a DNA/Fok I transducer. The transducer could cyclically cleave fluorescence-quenched probes to yield activated fluorescence signal, while protein binding to the small molecule label would prevent Fok I from approaching and cleaving the fluorescence-quenched probes. Because of the efficient signal amplification from the cyclic cleavage operation, the developed strategy could offer high sensitivity for detecting small molecule protein interactions. This strategy was demonstrated using folate and its high-affinity or low-affinity binding proteins. The results revealed that the developed strategy was highly sensitive for detecting either high- or low-affinity small molecule protein interactions with improved selectivity against nonspecific protein adsorption. This strategy could also be extended for assays of candidate small-molecule ligands using a competitive assay format. Moreover, this strategy only required labeling the small molecule on a DNA heteroduplex, circumventing protein modifications that might be harmful for activity. In view of these advantages, this new method could have potential to become a universal, sensitive, and selective platform for quantitative assays of small molecule protein interactions.