Fluorescent RNA Labeling and Inhibition Assay

All transcription reactions were performed with NTPs (Thermo Scientific; catalog #s: ATP – R0441, CTP – R0451, GTP – R0461, and UTP – R0471), 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) dye (Sigma Aldrich; catalog # SML1627), and RiboLock RNase inhibitors (Thermo Scientific; catalog # EO0381). DFHBI concentration, stored in 100 mM HEPES, pH 7.6, was determined by absorbance using an extinction coefficient at 420 nm of 31,611 M⁻¹cm⁻¹. Salmon-sperm DNA (Invitrogen; catalog # 15632011) was used as a competitor for single-round experiments (17 (link)). Rifampicin (Sigma-Aldrich; catalog # R3501) and Fidaxomicin (Selleckchem; catalog # S4227) solids were dissolved in DMSO. The molar concentration of Fidaxomicin was determined by weight and of Rifampicin by absorbance using an extinction coefficient at 470 nm of 15,300 M⁻¹cm⁻¹ (88 ).