Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
RNA. 2015 Feb;21(2):296-305. doi: 10.1261/rna.047159.114. Epub 2014 Dec 18.
A high-throughput assay for real-time measurement of translation rates in cell-free protein synthesis (SNAP assay) is described. The SNAP assay enables quantitative, real-time measurement of overall translation rates in vitro via the synthesis of O(6)-alkylguanine DNA O(6)-alkyltransferase (SNAP). SNAP production is continuously detected by fluorescence produced by the reaction of SNAP with a range of quenched fluorogenic substrates. The capabilities of the assay are exemplified by measurements of the activities of Escherichia coli MRE600 ribosomes and fluorescently labeled E. coli mutant ribosomes in the PURExpress translation system and by determination of the 50% inhibitory concentrations (IC50) of three common macrolide antibiotics.
本文描述了一种用于实时测量无细胞蛋白合成中翻译速率的高通量测定法（SNAP 测定法）。通过合成 O(6)-烷基鸟嘌呤 DNA O(6)-烷基转移酶（SNAP），SNAP 测定法可定量、实时测量体外的整体翻译速率。通过 SNAP 与一系列淬灭荧光底物的反应产生的荧光，连续检测 SNAP 的产生。该测定法的功能通过在 PURExpress 翻译系统中测量大肠杆菌 MRE600 核糖体和荧光标记的大肠杆菌突变核糖体的活性以及确定三种常见大环内酯抗生素的 50%抑制浓度（IC50）来举例说明。
