Cyclic enzymatic amplification method for highly sensitive detection of nuclear factor-kappa B

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Accurate detection of intracellular transcription factors is crucial for biological research and medical diagnostics.
• Nuclear factor-kappa B (NF-κB) is a key transcription factor involved in various cellular processes.

Purpose of the Study:

• To develop a rapid, simple, and highly sensitive fluorescence sensing strategy for detecting NF-κB.
• To establish a universal protocol for quantifying other DNA-binding proteins.

Main Methods:

• Utilized a fluorescence sensing strategy involving Exonuclease III (Exo III) digestion and TaqMan probes.
• Leveraged the sequence-specific binding of NF-κB to double-stranded DNA to protect one strand from Exo III digestion.
• Triggered an Exo III-mediated hydrolysis cycle on TaqMan probes for signal amplification.

Main Results:

• Achieved highly sensitive detection of NF-κB with a limit of detection as low as 45.6 pM.
• Demonstrated good selectivity due to the sequence-specific binding of NF-κB to DNA.
• Successfully quantified nuclear proteins directly from cell extracts.

Conclusions:

• The developed method provides a powerful tool for sensitive and selective NF-κB detection.
• The universal sensing protocol, adaptable for other DNA-binding proteins, has significant potential in disease diagnosis and pharmaceutical research.