Sensitive detection of transcription factor by coupled fluorescence-encoded microsphere with exonuclease protection

Aberrant transcription factors (TFs) activities are closely related to the occurrence and development of various diseases. Herein, we presented a fluorescence-encoded microsphere-based approach for TFs detection coupling with common DNA footprinting assay. Target TFs specifically bound the binding sites of double-stranded DNA (dsDNA) probes which were conjugated to microspheres. Thus, the probes were protected from being hydrolyzed by exonuclease III (Exo III). Afterwards, biotins labeled on the probes reacted with streptavidin-phycoerythrin (SA-PE) to produce fluorescent signal; however, in the absence of target TFs, the dsDNA probes would be hydrolyzed by Exo III resulting in biotins falling off and thus fluorescence signal was not generated. This strategy can be used to detect nuclear factor-kappa B p50 (NF-κB p50) with a detection limit of 0.2 nM. The steric hindrance of microspheres overcome the disadvantage of Exo III that can nibble into the protein-bound DNA region. Meanwhile, the fluorescent label of microsphere was specific to each TF, enabling multiplex detection could be achieved by changing specific protein binding site of corresponding dsDNA probe. This method has been successfully applied for simultaneous detection of NF-κB p50, AP-1 and CREB in nuclear extract isolated from HeLa cells stimulated or unstimulated by TNF-α, showing great potential for biomedical researches and precise disease diagnosis.