Highly sensitive detection of DNA-binding proteins based on a cationic conjugated polymervia a target-mediated fluorescence resonance energy transfer (TMFRET) strategy

Abstract

The sensitive, ratiometric and simple detection of sequence-specific DNA-binding proteins is of paramount importance for proteomics, genomics and biomedicine. We describe herein a novel target-mediated fluorescence resonance energy transfer (TMFRET) strategy for the homogeneous and visual detection of sequence-specific DNA-binding proteins. This conjugated polymer-based optical biosensor was demonstrated using a model target DNA-binding protein nuclear factor-kappaB (NF-κB), which is a key transcription factor involved in a number of important transcriptional regulatory networks. A fluorophore-labeled, double-stranded DNA probe bearing an NF-κB-binding site was designed to identify the target protein. Combining the protein with the probe could protect the probe from digestion by exonuclease III, yielding a high-efficiency FRET from the cationic conjugated polymer to the fluorophore label on the probe. The results revealed a highly sensitive, selective, colorimetric and turn-on detection of the purified recombinant NF-κB and HeLa cell nuclear extracts. The limit of detection for the target protein in nuclear extracts was estimated to be 1 × 10⁻⁵ μg μL⁻¹, which is 5000-fold more sensitive than previously reported methods. This strategy offers a new method for the reproducible, low-cost and simple detection of DNA-binding proteins because an average percent relative standard deviation of less than 5% was obtained. The results of the assay can also be seen by the naked eye with the help of a transilluminator, which is promising for detecting DNA-binding proteins in human samples for low-cost medical diagnoses and for the high-throughput screening of new drugs targeted to DNA-binding proteins.