滚动圆复制
DNA连接酶
生物标志物
DNA
连接酶连锁反应
生物
分子生物学
计算生物学
遗传学
聚合酶链反应
基因
DNA复制
多重聚合酶链反应
作者
Bing-Zhi Li,Bin Zhou,Yue Chen,Xing Zhang,He Huang
标识
DOI:10.1016/j.snb.2022.131598
摘要
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. • TFs-DNA binding can inhibit the approach between T4 DNA ligase and DNA nicks. • T4 DNA ligase is applied in the transducing of protein-DNA binding for the first time. • T4LiP accurately transduces TFs-DNA binding without sabotaging the TFs-DNA complex. • Sensitive detection of TFs is realized by integrating rolling circle amplification.
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