Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel, Anima Cell Metrology, Inc., Bernardsville, NJ 07924-2270, USA and Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA.
Nucleic Acids Res. 2013 Oct;41(18):e177. doi: 10.1093/nar/gkt686. Epub 2013 Aug 21.
The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Förster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the background transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2-Ibaraki and measured, through DiCoMPS, the synthesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fl-tRNA(Ile)UAU-generated FRET signals were specifically enhanced in infected cells, increased in the course of infection and were diminished on siRNA-mediated knockdown of NS3. Our results establish an experimental approach for the single-cell measurement of the levels of synthesis of a specific viral protein.
本报告进一步介绍了我们之前报道的技术，称为荧光转移 RNA（tRNA）用于翻译监测（FtTM），用于监测单个活细胞中活跃的全球蛋白质合成位点。FtTM 测量荧光 tRNA（fl-tRNA）占据核糖体上相邻位置时产生的Förster 共振能量转移（FRET）信号。当前的技术，称为蛋白质合成的二密码子监测（DiCoMPS），是为监测特定蛋白质的活跃合成而开发的。在 DiCoMPS 中，根据它们与感兴趣的 mRNA 中结合的二密码子序列的丰度相对于进行测定的细胞的背景转录组，选择特定的 fl-tRNA 对组合进行转染。在这项研究中，我们使用感染了伊巴拉克出血性疾病病毒 2 的细胞，并通过 DiCoMPS 测量了病毒非结构蛋白 3（NS3）的合成，该蛋白在 AUA:AUA 二密码子中富集。在感染细胞中，fl-tRNA（Ile）UAU 产生的 FRET 信号特异性增强，在感染过程中增加，并在 NS3 的 siRNA 介导的敲低时减少。我们的结果建立了一种用于单细胞测量特定病毒蛋白合成水平的实验方法。
