FRET-based protein-DNA binding assay for detection of active NF-kappa B

The Nuclear Factor kappa B (NF-kB), a transcription factor regulating a battery of inflammatory genes, has been implicated as playing a fundamental role in the development of numerous pathological states. The NF-kB dimer is located in the cytoplasm of cells in an inactive form that can be activated by many stimuli (e.g. cytokines, bacterial or viral products, UV-radiation, etc.). It has been demonstrated that anti-inflammatory agents, both steroid and non steroid, besides their principal mechanism of action, have a secondary mechanism with anti-NF-kB effects. Therefore, when anti-inflammatory therapy is applied to an inflammatory disease, for which detection of the efficacy of the therapeutic agent is particularly important, monitoring of NF-kB-active form concentration (for example in cellular lysate) is an important target. The critical need for a simple and direct method to evaluate the presence of active NF-kB in a biological sample can be addressed using a suitable and reusable biosensor. For this purpose, a novel method, using fluorescence resonance energy transfer (FRET), to detect the active form of NF-kB binding a specific DNA sequence has been developed. In order to evaluate FRET due to the DNA/protein binding interaction taking place between dsDNA immobilized in a capillary wall and p50 proteins, a highly sensitive FRET-based biosensor system developed in our laboratory was used.