Signal-amplified detection of biomarker transcription factors with improved accuracy based on T4 DNA ligase protection and rolling circle amplification

Transcription factors (TFs) are emerging tumor biomarkers that regulate gene expression. The detection of TFs depends on the transduction of TF-DNA binding to the nucleic acid signal, which mostly relies on exonuclease III (Exo III) protection assays. However, the processive digestion of Exo III usually causes undesired dissociation of the TF-DNA complex, resulting in low assay accuracy. We proposed a strategy, named T4 DNA ligase protection (T4LiP), to transduce the binding signal and then coupled it with rolling circle amplification (RCA) to fluorescently detect TFs. TFs bound to the nick region of dsDNA can prevent the association of T4 DNA ligase with the nick, thus inhibiting the ligation. Because of the mild activity of T4 DNA ligase, negligible dissociation of the TFs from dsDNA was observed. Using this to our advantage, we transduced the TF-DNA binding signal to generate circular DNA and then loaded it with RCA to sensitively detect p50 protein, yielding a detection limit of 0.5 pM. By sequence engineering, the detection of multiple TFs including MITF, p53, AP-1, and c-Myc with low picomolar sensitivities was accomplished. This is the first report of ligase-assisted transduction of TF-DNA binding, which shows high stability and versatility and holds the potential to improve the performance of TF assays.

Automated summary

In this study, a new strategy was proposed to sensitively detect transcription factors using T4 DNA ligase protection assay and rolling circle amplification. By preventing the ligation reaction through the binding of T4 DNA ligase to the nick region of dsDNA, the undesired dissociation of TF-DNA complexes was effectively avoided, improving the accuracy of the assay. Through sequence engineering, multiple transcription factors can be detected at low picomolar levels.