Protein binding protection in combination with DNA masking for sensitive and reliable transcription factor detection

Sensitive and reliable detection of transcription factors (TFs) is crucial for disease diagnosis and drug discovery. Herein, a protein binding protection in combination with DNA masking-based strategy is developed for sensitive and reliable detection of NF-κB p50. First, NF-κB p50 binds specifically to a hairpin probe to form a protein-DNA complex. Then, the formed complex protects the hairpin probe from hybridization with the masking strand and subsequently triggers the cascade signal amplification of strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). Excess hairpin probes that do not bind NF-κB p50 are masked through hybridization with masking strands into stable duplexes, prohibiting the non-specific amplification and avoiding the risk of false positive signals. The signal results from the presence of NF-κB p50, ensuring the detection reliability. Through the cascade amplification specifically triggered by the target, the method can detect the purified recombinant NF-κB p50 down to 1.0 × 10^-13 M. It is further employed for the NF-κB p50 inhibitor screening and analysis, which shows the potential in anti-NF-κB p50 drug discovery. Moreover, the method is applied in detection NF-κB p50 in cell nuclear extracts with a detection limit of 0.1 ng μL^-1. The proposed strategy will provide a promising tool for sensitive and reliable assaying NF-κB p50 activity in biomedical study and disease diagnosis.