Amplified detection of nuclear factor-kappa B activity and inhibition based on exonuclease III assisted cleavage-induced DNAzyme releasing strategy

The transcription factors (TFs) plays an essential role in the cellular components to regulate gene expression, gene replication, gene repair, gene transcription and cell division. In this study, we proposed a simple, rapid and selective fluorescent method for monitoring TFs activity based on a exonuclease III (Exo III) cleavage-induced DNAzyme releasing strategy. A double-stranded DNA (dsDNA) which contains TFs binding site and 8-17 DNAzyme units was designed. With the addition of nuclear factor-kappa B p50 (NF-κB p50), the digestion of Exo III cannot proceed past the binding site, then the 8-17 DNAzyme substrate was successfully released and show its cyclic catalytic cleavage toward molecular beacon (MB) substrate, resulting in an evident fluorescence signal enhancement. With this currently developed Exo III cleavage reaction and DNAzyme-based platform, a detection limit of 6.2 pM could be achieved. Moreover, the inhibition effects of oridonin on NF-κB p50 have also been evaluated with satisfactory results. This developed Exo III cleavage-induced DNAzyme releasing strategy opens a promising avenue for monitoring activity and inhibition of nucleotide kinase, and should be also conveniently used for the sensor of other proteins or nucleic acid and may have widespread applications in biological process researches, drug discovery, and clinic diagnostics.