Persia for Direct Fluorescence Measurements of Transcription,Translation, and Enzyme Activity in Cell-Free Systems

In a cell-free reaction, we would like to know what is happening and when it is happening. We employ an approach we term PERSIA (for PureExpress-ReAsH-Spinach-In-vitro-Analysis) that allows us to observe the core processes of cell-free transcription and translation during the reaction. We combine small molecule labels (DFHBI and ReAsH) with RNA and peptide tags (Spinach and tetracysteine) that produce fluorescent signals upon binding reporting on the synthesis of the full-length biomolecule. Avoiding GFP and other fluorescent proteins allows us to measure production directly for our many different proteins of interest. Coupled with de novo DNA synthesis, we have applied PERSIA to answering several different kinds of questions, such as the practicability of designed genetic codes, scanning mutagenesis of full length proteins, and probing drug resistance mutations. Additional opportunities with PERSIA remain such as optimizing protein yields for heterologous expression.