Transcription factors are involved in a number of important cellular processes. The
transcription factor NF-κB has been linked with a number of
cancers, autoimmune and inflammatory diseases. As a result, monitoring
transcription factors potentially represents a means for the early detection and prevention of diseases. Most methods for
transcription factor detection tend to be tedious and laborious and involve complicated sample preparation, and are not practical for routine detection. We describe herein the first label-free luminescence switch-on detection method for
transcription factor activity using
Exonuclease III and a luminescent
ruthenium complex, [
Ru(phen)(2)(dppz)](2+). As a proof of concept for this novel assay, we have designed a
double-stranded DNA sequence bearing two NF-κB binding sites. The results show that the luminescence response was proportional to the concentration of the NF-κB subunit p50 present in the sample within a wide concentration range, with a nanomolar detection limit. In the presence of a known NF-κB inhibitor,
oridonin, a reduction in the luminescence response of the
ruthenium complex was observed. The reduced luminescence response of the
ruthenium complex in the presence of small molecule inhibitors allows the assay to be applied to the high-throughput screening of
chemical libraries to identify new antagonists of
transcription factor DNA binding activity. This will allow the rapid and low cost identification and development of novel scaffolds for the treatment of diseases caused by the deregulation of
transcription factor activity.