Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland.
College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland.
Chemistry. 2019 May 10;25(27):6728-6740. doi: 10.1002/chem.201900051. Epub 2019 Apr 1.
The m G cap is a unique nucleotide structure at the 5'-end of all eukaryotic mRNAs. The cap specifically interacts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecular probes to study important cap-recognizing proteins, we synthesized m G nucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymatic cleavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. A pyrene-labeled m GTP analogue showed up to eightfold enhanced fluorescence emission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-fold enhancement upon cleavage by decapping scavenger (DcpS) enzyme. These observations served as the basis for developing binding- and hydrolytic-activity assays. The assay utility was validated with previously characterized libraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also applied to study hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells.
mG 帽是所有真核生物 mRNA 5' 端的一种独特核苷酸结构。该帽结构特异性地与许多细胞蛋白相互作用，并参与对细胞生长和功能至关重要的生物过程。为了提供小分子探针来研究重要的帽识别蛋白，我们通过末端磷酸（膦）酸基团合成了带有荧光标记的 mG 核苷酸，并研究了它们的发射特性在与蛋白结合或酶切时如何发生变化。只有芘标记的化合物表现为灵敏的开启型探针。当与真核翻译起始因子 4E (eIF4E) 结合时，芘标记的 mGTP 类似物的荧光发射强度增强了 8 倍以上，当被脱帽清除酶 (DcpS) 酶切割时，增强了 30 多倍。这些观察结果为开发结合和水解活性测定奠定了基础。该测定方法的有效性已通过先前表征的 eIF4E 配体和 DcpS 抑制剂文库得到验证。该 DcpS 测定法还应用于研究细胞质提取物中内源性酶的水解活性和抑制作用，这些提取物来自 HeLa 和 HEK 细胞。
