Fluorogenic RNA Aptamer-Based Activity Quantification of T7 RNA Polymerase

T7 RNA polymerase (T7 RNAP) is a central molecular biology tool that is broadly used for biomedical research and the RNA therapeutic industry. T7 RNAP and its engineered variants provide valuable tools in synthetic biology and gene expression regulation. T7 RNAP activity unit is traditionally quantified by a radioactivity-based polymerization assay, which is often not accessible to many laboratories and raises safety concerns because of exposure to radioactivity. Here, we developed a fluorescence-based in vitro transcription (IVT) assay that enables facile and quantitative determination of T7 RNAP activity units, using the Broccoli RNA aptamer as a transcriptional reporter and commercially available T7 RNAPs as standard references, which are readily accessible materials for most biochemical laboratories. This method will improve the consistency and reproducibility assessment of T7 RNAP prepared from different batches or different laboratories and benefit research communities that rely on the robust performance of RNA polymerases.