Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 200 First St SW, Rochester, Minnesota 55905, USA.
Department of Biological Engineering, Massachusetts Institute of Technology, Boston, MA 02139, USA.
Chembiochem. 2021 Apr 16;22(8):1400-1404. doi: 10.1002/cbic.202000854. Epub 2021 Jan 27.
Recent advances in peroxidase-mediated biotin tyramide (BT) signal amplification technology have resulted in high-resolution and subcellular compartment-specific mapping of protein and RNA localization. Horseradish peroxidase (HRP) in the presence of H O is known to activate phenolic compounds for phenoxy radical reaction with nucleic acids, where biotinylation by BT is a practical example. BT reactivity with RNA and DNA is not understood in detail. We report that BT phenoxy radicals react in a sequence-independent manner with guanosine bases in RNA. In contrast, DNA reactivity with BT cannot be detected by our methods under the same conditions. Remarkably, we show that fluorescein conjugates DNA rapidly and selectively reacts with BT phenoxy radicals, allowing convenient and practical biotinylation of DNA on fluorescein with retention of fluorescence.
近年来，过氧化物酶介导的生物素酪胺（BT）信号放大技术的进展使得蛋白质和 RNA 定位的高分辨率和亚细胞区室特异性作图成为可能。众所周知，辣根过氧化物酶（HRP）在 H2O 的存在下会激活酚类化合物，使其与核酸发生苯氧基自由基反应，其中 BT 的生物素化就是一个实际的例子。BT 与 RNA 和 DNA 的反应机制尚不清楚。我们报告称，BT 苯氧基自由基以序列非依赖性的方式与 RNA 中的鸟嘌呤碱基反应。相比之下，在相同条件下，我们的方法无法检测到 DNA 与 BT 的反应。值得注意的是，我们表明荧光素缀合的 DNA 可快速且选择性地与 BT 苯氧基自由基反应，从而可以方便、实用地在荧光素上对 DNA 进行生物素化，同时保留荧光。
