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

Louis Kuijpers; Theo van Laar; Richard Janissen; Nynke H Dekker

doi:10.1016/j.xpro.2022.101606

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

STAR Protoc. 2022 Aug 5;3(3):101606. doi: 10.1016/j.xpro.2022.101606. eCollection 2022 Sep 16.

Authors

Louis Kuijpers¹, Theo van Laar¹, Richard Janissen², Nynke H Dekker³

Affiliations

¹ Department of Bionanoscience, Delft University of Technology, 2629 HZ Delft, the Netherlands.
² Department of Bionanoscience, Delft University of Technology, 2629 HZ Delft, the Netherlands. Electronic address: r.janissen@tudelft.nl.
³ Department of Bionanoscience, Delft University of Technology, 2629 HZ Delft, the Netherlands. Electronic address: n.h.dekker@tudelft.nl.

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).

Keywords: Biophysics; Microbiology; Molecular biology; Molecular/Chemical probes; Single-molecule assays.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antiviral Agents*
Kinetics
Magnetic Phenomena
RNA, Double-Stranded
RNA-Dependent RNA Polymerase*

Substances

Antiviral Agents
RNA, Double-Stranded
RNA-Dependent RNA Polymerase