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

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

Research output: Contribution to journalArticlepeer-review

42 Scopus citations


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 platform to monitor the elongation dynamics of a prototypical RdRp over thousands of nucleotide-addition cycles in the absence and presence of a suite of nucleotide analog inhibitors. We observe multiple RdRp-RNA 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. Dulin et al. find that a prototypical RNA-dependent RNA polymerase (RdRp) visits several states during nucleotide synthesis, of which only one incorporates nucleotide analogs with therapeutic potential. Different analogs exhibit distinct kinetic signatures, with an analog thought to induce chain termination actually promoting RdRp backtracking.

Original languageEnglish (US)
Pages (from-to)1063-1076
Number of pages14
JournalCell reports
Issue number4
StatePublished - Oct 24 2017

Bibliographical note

Publisher Copyright:
© 2017 The Authors


  • RNA polymerase
  • RNA virus
  • T-1106
  • T-705
  • backtracking
  • inhibitor
  • magnetic tweezers
  • nucleoside analog
  • pyrazine carboxamide


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