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

Effective detection of the intracellular expression level of transcription factors is important for biological research and medical diagnosis. This study proposes a rapid and simple fluorescence sensing strategy for highly sensitive detection of a transcription factor, nuclear factor-kappa B (NF-kappa B). NF-kappa B binds to double-stranded DNA duplex, one of which is then protected from Exonuclease III (Exo III) digestion. An Exo III-mediated hydrolysis cycle on TaqMan probes is then triggered to achieve highly sensitive detection of NF-kappa B. This method can detect NF-kappa B with concentration as low as 45.6 pM. Furthermore, sequence-specific binding of NF-kappa B to DNA provides good selectivity. This method can be used for the direct quantification of nuclear proteins extracted from cells. More importantly, by simply replacing the sequence of the probe binding site, this method can also be used for reliable quantification of other DNA-binding proteins and is thus a universal sensing protocol. This strategy can be a powerful technology in the areas of disease diagnosis and pharmaceutical research. (C) 2019 Elsevier B.V. All rights reserved.