T7 RNA Polymerase (RNAP) is a widely used enzyme with recent applications in the production of RNA vaccines. For over 50 years denaturing sequencing gels have been used as key analysis tools for probing the nucleotide addition mechanisms of T7 RNAP and other polymerases. However, sequencing gels are low-throughput limiting their utility for comprehensive enzyme analysis. Here, we report the development of HiKER; (High-throughput Kinetics using Capillary Electrophoresis and Robotics) a high-throughput pipeline to quantitatively measure enzyme kinetics. We adapted a traditional polymerase misincorporation assay for fluorescent detection at scale allowing rapid estimates of RNAP misincorporation in different experimental conditions. Using this platform with an OT-2 robotics system, ~1500 time points were collected in a single workday. T7 RNAP exhibited dramatic differences in both observed rate constant and amplitude depending on the mismatch examined. An average misincorporation frequency of ~45 misincorporations per million bases was estimated using HiKER and is consistent with previous studies. Misincorporation time courses for T3 RNAP and Sp6 RNAP were also collected and appeared similar to T7 RNAP suggesting conserved kinetic mechanisms. However, differences between the RNAPs were observed in extension from base mismatch experiments. This platform is affordable, open-source, and broadly applicable to many enzymes.
Copyright: © 2024 Carter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.