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Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA
Peter D Leitner1,2,3, Ilja Vietor1, Lukas A Huber1,2
1Institute of Cell Biology, Biocenter, Medical University of Innsbruck, Innrain 80-82, 6020, Innsbruck, Austria.
Abstract:
The nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1-306) and p50 (aa 1-367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (KD) of 2.228 × 10-6 M and 0.794 × 10-6 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC) verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.
Insights
A new fluorescence assay quantifies nuclear factor kappa B (NF-κB) DNA binding by measuring protein thermal stability. This method aids in studying NF-κB activity and screening for drugs targeting cancers and inflammatory diseases.
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 (KD) 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.
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