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