Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA

Area of Science:

• Molecular Biology
• Biochemistry
• Drug Discovery

Background:

• Nuclear factor kappa B (NF-κB) is a transcription factor family regulating genes involved in cancer, autoimmunity, and inflammation.
• Effective high-throughput methods are crucial for studying NF-κB transcriptional activity and for screening potential inhibitory drugs.
• Current methods may not fully capture the nuances of NF-κB DNA binding kinetics and drug interactions.

Purpose of the Study:

• To develop and validate a novel fluorescence-based assay for quantitative detection of NF-κB DNA binding.
• To assess the assay's utility in drug screening by measuring dose-dependent inhibitory effects on NF-κB binding.
• To enable the analysis of direct inhibitor binding and its impact on protein structural stability.

Main Methods:

• Designed NF-κB protein probes (p65/RelA and p50) using bioinformatic analysis.
• Employed SYPRO Orange fluorescence to monitor thermal denaturation and measure shifts in melting temperatures (ΔTm) upon DNA binding.
• Validated the assay using known NF-κB inhibitors: withaferin A (WFA), dimethyl fumarate (DMF), and p-xyleneselenocyanate (p-XSC).

Main Results:

• The assay successfully detected and quantified dsDNA binding by measuring the thermal stability shift (ΔTm) of NF-κB protein probes.
• The increase in melting temperature was proportional to dsDNA concentration, yielding apparent dissociation constants (K_D) for p65/RelA and p50.
• The assay effectively measured dose-dependent inhibition of NF-κB DNA binding by WFA, DMF, and p-XSC, confirming its suitability for drug screening.

Conclusions:

• A novel, quantitative fluorescence-based assay accurately measures NF-κB DNA binding through protein thermal stability.
• This assay is valuable for studying NF-κB transcriptional activity, screening for novel therapeutic agents, and understanding drug-target interactions.
• The method facilitates the identification and rational design of drug candidates that interfere with NF-κB signaling pathways.