Amplified probing of protein/DNA interactions for sensitive fluorescence detection of transcription factors

Abstract

Protein/DNA interactions play important roles in many cellular activities, and sensitive monitoring of such interactions can potentially elucidate the regulation of gene expression. In this work, on the basis of specific protein/DNA interactions, we describe the establishment of a simple and sensitive method for detecting transcription factors by coupling the nuclease protection strategy with metal-ion dependent DNAzyme amplification. Nuclear factor κB (NF-κB) p50 target molecules specifically bind DNAzymes containing DNA duplex probes and protect them from being digested by exonuclease III. Consequently, the protected DNAzymes react with the fluorescently quenched substrate hairpin signal probes and cause cyclic cleavage of the signal probes to generate substantially amplified fluorescent readouts for sensitively detecting NF-κB p50. Our method exhibits a dynamic range of 0.01 to 50 nM and a detection limit of 0.54 pM, respectively, for NF-κB p50. Moreover, due to the specific interaction between the target protein and the duplex probes, high selectivity towards NF-κB p50 against other control proteins is observed. With the features of simplicity, sensitivity and selectivity, the developed method can thus be employed to monitor different types of protein/DNA interactions for biological studies and disease diagnosis.