Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA 
Polymerase Revealed Using High-Throughput Magnetic Tweezers

Dulin, David; Arnold, Jamie J.; van Laar, Theo; Oh, Hyung-Suk; Lee, Cheri; Perkins, Angela L.; Harki, Daniel A.; Depken, Martin; Cameron, Craig E.; Dekker, Nynke H.

doi:10.1016/j.celrep.2017.10.005

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Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers

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Dulin, David
Max-Planck-Zentrum für Physik und Medizin, Max Planck Institute for the Science of Light, Max Planck Society;
Delft University of Technology;
Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU);

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Citation

Dulin, D., Arnold, J. J., van Laar, T., Oh, H.-S., Lee, C., Perkins, A. L., et al. (2017). Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers. Cell Reports, 21(4), 1063-1076. doi:10.1016/j.celrep.2017.10.005.

Cite as: https://hdl.handle.net/21.11116/0000-0006-AFE3-5

Abstract

RNA viruses pose a threat to public health that is exacerbated by the dearth of antiviral therapeutics. The RNA-dependent RNA polymerase (RdRp) holds promise as a broad-spectrum, therapeutic target because of the conserved nature of the nucleotide-substrate-binding and catalytic sites. Conventional, quantitative, kinetic analysis of antiviral ribonucleotides monitors one or a few incorporation events. Here, we use a high-throughput magnetic tweezers platformto monitor the elongation dynamics of a prototypicalRdRpover thousands of nucleotide-addition cycles in the absence and presence of a suite of nucleotide analog inhibitors. We observe multiple RdRpRNA elongation complexes; only a subset of which are competent for analog utilization. Incorporation of a pyrazine-carboxamide nucleotide analog, T-1106, leads to RdRp backtracking. This analysis reveals a mechanism of action for this antiviral ribonucleotide that is corroborated by cellular studies. We propose that induced backtracking represents a distinct mechanistic class of antiviral ribonucleotides.