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

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

40 Citations (Scopus)
60 Downloads (Pure)

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 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
Pages (from-to)1063-1076
Number of pages14
JournalCell Reports
Volume21
Issue number4
DOIs
Publication statusPublished - 2017

Keywords

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

Fingerprint

Dive into the research topics of 'Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers'. Together they form a unique fingerprint.

Cite this