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
Affiliations
David Dulin
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands; Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich Alexander University Erlangen-Nürnberg (FAU), Hartmannstr. 14, 91052 Erlangen, Germany
Jamie J. Arnold
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
Theo van Laar
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands
Hyung-Suk Oh
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
Cheri Lee
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
Angela L. Perkins
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
Daniel A. Harki
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
Martin Depken
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands; Corresponding author
Craig E. Cameron
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA; Corresponding author
Nynke H. Dekker
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands; Corresponding author
Summary: 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. Keywords: RNA virus, RNA polymerase, magnetic tweezers, inhibitor, nucleoside analog, backtracking, pyrazine carboxamide, T-1106, T-705
