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

• Published in: ACS synthetic biology 2019-05, Vol.8 (5), p.1010-1025
• Main Authors: Wick, Scott, Walsh, David I, Bobrow, Johanna, Hamad-Schifferli, Kimberly, Kong, David S, Thorsen, Todd, Mroszczyk, Keri, Carr, Peter A
• Format: Article
• Language: English
• Subjects: Cell-Free System; Genetic Engineering - methods; HIV - enzymology; HIV Protease - genetics; HIV Protease - metabolism; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Plant Proteins - genetics; Plant Proteins - metabolism; Protein Biosynthesis; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; RNA, Messenger - metabolism; Spectrometry, Fluorescence; Spinacia oleracea - genetics; Transcription, Genetic; Ubiquitin - genetics; Ubiquitin - metabolism
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.8b00450

Quantification of biology’s central dogma (transcription and translation) is pursued by a variety of methods. Direct, immediate, and ongoing quantification of these events is difficult to achieve. Common practice is to use fluorescent or luminescent proteins to report indirectly on prior cellular events, such as turning on a gene in a genetic circuit. We present an alternative approach, PURExpress-ReAsH-Spinach In-vitro Analysis (PERSIA). PERSIA provides information on the production of RNA and protein during cell-free reactions by employing short RNA and peptide tags. Upon synthesis, these tags yield quantifiable fluorescent signal without interfering with other biochemical events. We demonstrate the applicability of PERSIA in measuring cell-free transcription, translation, and other enzymatic activity in a variety of applications: from sequence–structure–function studies, to genetic code engineering, to testing antiviral drug resistance.