Characterizing single-molecule dynamics of viral RNA-dependent RNA polymerases with multiplexed magnetic tweezers
L.C. Kuijpers (TU Delft - BN/Nynke Dekker Lab)
T. van Laar (TU Delft - BN/Nynke Dekker Lab)
R. Janissen (TU Delft - BN/Cees Dekker Lab)
N.H. Dekker (TU Delft - BN/Nynke Dekker Lab)
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Abstract
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).
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