Platform for measuring polymerase nucleotide misincorporation kinetics reveals mechanistic differences between wild-type and engineered RNAPs

摘要: The 99-kDa single subunit enzyme, T7 RNA polymerase (RNAP), is a well-studied enzyme with studies dating back to the 1970’s. Historically, denaturing sequencing gels have been used as a key tool for probing the kinetic mechanism of T7 RNAP. Sequencing gels are both slow and low throughput making it challenging to probe many different experimental conditions. Here we have adapted a traditional T7 RNA polymerase misincorporation assay for fluorescent detection at scale allowing fidelity for fast estimates of RNAP fidelity in different experimental conditions. We report the development of NEB Tracer; a high-throughput pipeline to measure single nucleotide incorporation kinetics. Time courses monitoring nucleotide addition were generated using transient-state kinetic techniques in combination with a high-throughput capillary electrophoresis system. This experimental methodology allowed 20+ time …