Characterizing single-molecule dynamics of viral RNA-dependent RNA polymerases with multiplexed magnetic tweezers

Abstract

Summary: Multiplexed single-molecule magnetic tweezers (MT) have recently been employed to probe the RNA synthesis dynamics of RNA-dependent RNA polymerases (RdRp). Here, we present a protocol for simultaneously probing the RNA synthesis dynamics of hundreds of single polymerases with MT. We describe the preparation of a dsRNA construct for probing single RdRp kinetics. We then detail the measurement of RdRp RNA synthesis kinetics using MT. The protocol is suitable for high-throughput probing of RdRp-targeting antiviral compounds for mechanistic function and efficacy.For complete details on the use and execution of this protocol, please refer to Janissen et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Biophysics
• Microbiology
• Molecular biology
• Molecular/Chemical probes
• Single-molecule assays