Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B

Area of Science:

• Biochemistry
• Molecular Biology
• Biosensor Technology

Background:

• Nuclear Factor kappa B (NF-κB) is a key regulator of inflammatory responses and cell proliferation, making it a therapeutic target for cancer and chronic inflammatory diseases.
• Accurate detection of NF-κB activity is crucial for understanding disease mechanisms and developing targeted therapies.

Purpose of the Study:

• To develop and validate a novel molecular binding scheme for the sensitive and specific detection of NF-κB.
• To establish a quantitative, high-throughput, and cost-effective method for NF-κB measurement.

Main Methods:

• A molecular probe biosensor utilizing dye and quencher fluorophores was designed to detect NF-κB binding to Ig-κB DNA.
• Specificity was confirmed through competitive binding assays.
• A normalization equation was developed to correct for variations in fluorophore concentration and background noise.
• Periodic shaking was employed to stabilize DNA-protein binding.

Main Results:

• The biosensor demonstrated a detection limit in the nanomolar range for purified p50 (NF-κB).
• Specificity experiments revealed differential binding affinities: p50/p65 heterodimer showed the highest affinity, followed by p65 homodimer, and then p50 homodimer.
• TNF-α stimulation significantly increased the affinity of HeLa nuclear extract to Ig-κB DNA compared to unstimulated extract.

Conclusions:

• The developed molecular binding scheme provides a rapid, quantitative, and automated method for measuring DNA-binding protein NF-κB.
• This biosensor is suitable for high-throughput screening and automated drug discovery systems targeting NF-κB.
• The findings highlight the potential of this biosensor for applications in diagnostics and therapeutic development.