A simple real-time assay for in vitro translation

作者：Capece Mark C; Kornberg Guy L; Petrov Alexey; Puglisi Joseph D^*

来源：RNA-A Publication of the RNA Society, 2015, 21(2): 296-305.

DOI：10.1261/rna.047159.114

• SNAP
• in vitro translation
• assay
• single-molecule fluorescence
• IC50
• ribosome activity
• translation rate

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.

• 出版日期2015-2