Development of Selective, Label-Free, Homogenous Luminescent Switch-On Probe for the Detection of Nanomolar Transcription Factor

Transcription factors are one of the largest classes of DNA-binding proteins that regulate gene expression by binding to specific DNA sequences within the regulatory regions of genes, thereby controlling essential biological processes related to cell development, differentiation and growth. Certain transcription factors have the ability to regulate an entire subset of genes and thus their activity is tightly regulated under normal physiological conditions. Aberrant transcription factor activity has been associated with the development of a number of diseases including cancer, developmental disorders and inflammation. Advances in the biomedical sciences have validated transcription factors as disease markers and potential targets for therapeutic intervention. Consequently, there is an urgent need for the development of simple and economical methods for the detection of transcription factors in biological systems.

Traditional approaches for protein detection are time-consuming, expensive, or involve radiographic techniques. In this proposal, we aim to develop an oligonucleotide-based, label-free luminescent detection method for transcription factors using structure- specific transition metal complexes that can transduce the structure-switching response of the oligonucleotide into a luminescence response. Our preliminary results have demonstrated that a ruthenium complex can function as a luminescent probe to monitor the structure-switching response of an unlabeled oligonucleotide in response to the transcription factor NF-κB.

We will build our preliminary results by rationally designing and synthesizing a series of new platinum(II), iridium(III) and rhodium(III) complexes as selective luminescent probes for G-quadruplex or duplex DNA using a combination of molecular modeling, biochemical and biophysical techniques. We will investigate several oligonucleotide designs and rigorously optimize the DNA sequence in order to maximize the selectivity, sensitivity and response time of our assay. We envision that the long phosphorescence lifetimes of transition metal complexes can be exploited for the in vitro detection of transcription factors in highly fluorescent biological media such as human cellular and nuclear extracts by use of time-resolved luminescence spectroscopy. Our proposed methodology is highly simple, sensitive, rapid, selective, low-cost and amenable to real- time and high-throughput analysis. We anticipate that the proposed technology could be highly useful to both academic and biomedical researchers worldwide working in the field of transcription factor biochemistry, and could be potentially applied for the clinical diagnosis or treatment of human diseases.