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BP: Fachverband Biologische Physik

BP 19: Poster VII

BP 19.19: Poster

Dienstag, 17. März 2020, 14:00–16:00, P2/3OG

Temperature controlled high-throughput magnetic tweezers assay for viral RNA-dependent RNA polymerase study — •Mona Seifert¹, Pauline van Nies¹, Flávia Stal Papini¹, Jamie Arnold², Minna Poranen³, Craig Cameron², Martin Depken⁴, and David Dulin¹ — ¹IZKF, FAU Erlangen-Nürnberg — ²The University of North Carolina Chapel Hill — ³University of Helsinki — ⁴TU Delft

The viral RNA-dependent RNA polymerase (RdRp) is an essential factor for the virus to establish a successful infection as it generates all viral RNA. As enzymatic kinetic processes follow the Arrhenius law, polymerase nucleotide addition rate is expected to be temperature sensitive. We previously introduced high-throughput magnetic tweezers to study RdRp kinetics using kilobases long templates, i.e. a length similar to the viral genome, with near single base resolution. To perform experiments at in vivo temperature, we developed a temperature controlling system for our magnetic tweezers assay and performed in situ temperature calibration by leveraging the temperature dependence of DNA twist. We applied the temperature controlled setup to study the elongation kinetics of different RdRps at several temperatures and we observed that the increase in temperature correlates with a higher nucleotide addition rate and short pause exit rate, confirming the catalytic nature of these pauses. Non-catalytic backtrack pauses however are temperature insensitive. The assay we present here simultaneously provides high throughput and temperature control, which will be essential for future studies of complex viral replicases.