Unimolecular beacons for the detection of DNA-binding proteins

A new methodology for detecting sequence-specific DNA-binding proteins has been recently developed (Heyduk, T.; Heyduk, E. Nat. Biotechnol. 2002, 20, 171). The core feature of this methodology is protein-dependent association of two fluorochrome-labeled DNA fragments, which allows generation of a fluorescence signal reporting the presence of the target protein. Previous kinetic experiments identified the association of the two DNA fragments as the rate-limiting step of the assay. Here we report on a variant of the assay, in which components of the assay-fluorescent DNA fragments-were covalently tethered by a non-DNA linker with the goal of increasing the rate of association of the two fragments. We investigated the effect of the tether on the performance of the assay under a variety of conditions using a model DNA-binding protein. Quantitative titrations and rapid kinetic stopped-flow experiments were conducted to validate the molecular model that describes the two linked equilibria: oscillation of the tethered construct between the open and closed states and the exclusive association of the protein with the closed state. Experiments were also performed to demonstrate the ability of these tethered constructs to signal when attached to a solid surface. The major advantage of this new assay format is the faster response time for the detection allowing the higher throughput of the analysis. Additionally, it will be possible to attach tethered beacons to other solid surfaces, thus allowing the preparation of arrays containing molecular beacons for many different DNA-binding proteins.