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<p>Nsp15 is an endoribonuclease, highly conserved among coronaviruses, that helps the virus evade detection in host cells by cleaving uridine-rich viral RNA sequences. Those sequences would otherwise trigger immune response pathways. Its essential role in pathogenesis and highly conserved nature make Nsp15 an attractive target for therapeutic intervention. While its crystal structure and uridine specificity are well-established, the influence of RNA structural dynamics and divalent cations on Nsp15 activity remains less understood. Leveraging single-molecule Pulsed Interleaved Excitation (PIE)-FRET and Fluorescence Correlation Spectroscopy (FCS) in combination, we developed an assay to track RNA cleavage by Nsp15 variants in real time and monitor the conformational dynamics of hybrid RNA/DNA substrates. Using our methodology and analysis strategies, we obtained clear indicators of RNA cleavage with both PIE-FRET and FCS data analysis. Our assay also revealed signatures of unique dynamic behavior in uridine-containing RNA substrates, indicating that divalent cations enhance substrate flexibility, which is associated with a faster observed reaction rate of RNA cleavage by Nsp15 in the presence of Mn<sup>2+</sup>. We propose that divalent cations induce a conformation that may be more favorable for cleavage in single-stranded RNA substrates that mediate the efficient nuclease activity of Nsp15.</p>

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A combined PIE-FRET and FCS assay to monitor RNA dynamics and cleavage by SARS-CoV-2 Nsp15

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月29日

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  • Kenya Gordon,
  • Zoe M. Wright,
  • Cameron R. Stephens,
  • Meredith N. Frazier,
  • Benjamin S. Clark,
  • Isha M. Wilson,
  • Irene Silvernail,
  • Robin E. Stanley,
  • Sharonda J. LeBlanc

摘要

Nsp15 is an endoribonuclease, highly conserved among coronaviruses, that helps the virus evade detection in host cells by cleaving uridine-rich viral RNA sequences. Those sequences would otherwise trigger immune response pathways. Its essential role in pathogenesis and highly conserved nature make Nsp15 an attractive target for therapeutic intervention. While its crystal structure and uridine specificity are well-established, the influence of RNA structural dynamics and divalent cations on Nsp15 activity remains less understood. Leveraging single-molecule Pulsed Interleaved Excitation (PIE)-FRET and Fluorescence Correlation Spectroscopy (FCS) in combination, we developed an assay to track RNA cleavage by Nsp15 variants in real time and monitor the conformational dynamics of hybrid RNA/DNA substrates. Using our methodology and analysis strategies, we obtained clear indicators of RNA cleavage with both PIE-FRET and FCS data analysis. Our assay also revealed signatures of unique dynamic behavior in uridine-containing RNA substrates, indicating that divalent cations enhance substrate flexibility, which is associated with a faster observed reaction rate of RNA cleavage by Nsp15 in the presence of Mn2+. We propose that divalent cations induce a conformation that may be more favorable for cleavage in single-stranded RNA substrates that mediate the efficient nuclease activity of Nsp15.