Molecular beacons for detecting DNA binding proteins: mechanism of action.

New methodology for detecting sequence-specific DNA binding proteins has been recently developed (T. Heyduk, and E. Heyduk, Nat. Biotechnol. 20 (2002) 171). The central feature of this assay is protein-dependent association of two DNA fragments, each containing about half of a DNA sequence-defining the protein binding site. In this report we propose a physical model explaining the functioning of the assay. The model involves two linked equilibria: association between the two DNA fragments and binding of the protein exclusively to the complex between the two DNA fragments. Equilibrium and kinetic experiments provided evidence supporting the proposed model and showed that the model was sufficient to describe the behavior of the assay under a variety of conditions. Kinetic data identified the association between the two DNA half-sites as the rate-limiting step of the assay. Theoretical simulations based on the proposed model were used to investigate parameters important for the maximal sensitivity of the assay. Physical understanding of the assay will provide means for rational design of the assay for a variety of target proteins.