NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, 214063, China; Department of Radiopharmaceuticals, School of Pharmacy, Nanjing Medical University, Nanjing, 211166, China.
NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, 214063, China; Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing, 211816, PR China.
Anal Chim Acta. 2020 Apr 1;1104:132-139. doi: 10.1016/j.aca.2019.12.076. Epub 2020 Jan 3.
Herein, we report a new probe for the determination of the concentration of NF-κB p50, one kind of DNA-binding transcription factors (TFs), by using Exonuclease III (Exo III)-aided amplification and gold nanoparticle mediated fluorescence intensity. Since TFs play critical roles in various biological processes, the detection of TFs can provide a lot of useful biological information for studding gene expression regulation related disease. In our system, in the presence of transcription factor, Exo III based amplification reaction was trigged. This enzymatic digestion results in the release of intermediate DNA and ultimately liberating the fluorophore (which, separated from the quencher of AuNP and BHQ2, now fluoresces). The released intermediate DNA then hybridizes with another strand3, whence the cycle starts anew. So, the fluorescence intensity reflects the NF-κB p50 concentration with a detection limit of 1.32 pM. Importantly, this method might be further extended to selectively detect various dsDNA-binding proteins by simply changing the binding-site sequences of strand1/strand2 duplex probes.
在这里，我们报告了一种新的探针，用于通过 Exonuclease III（Exo III）辅助扩增和金纳米粒子介导的荧光强度来测定 NF-κB p50 的浓度，NF-κB p50 是一种 DNA 结合转录因子（TFs）。由于 TFs 在各种生物过程中起着关键作用，因此 TFs 的检测可以为研究与基因表达调控相关的疾病提供大量有用的生物学信息。在我们的系统中，在转录因子存在的情况下，触发了基于 Exo III 的扩增反应。这种酶促消化导致中间 DNA 的释放，最终释放荧光团（与 AuNP 和 BHQ2 的猝灭剂分离，现在发出荧光）。释放的中间 DNA 然后与另一条链 3 杂交，循环重新开始。因此，荧光强度反映了 NF-κB p50 的浓度，检测限为 1.32 pM。重要的是，通过简单地改变链 1/链 2 双链探针的结合位点序列，该方法可能进一步扩展到选择性检测各种 dsDNA 结合蛋白。
