Poster abstracts

Poster number 63 submitted by Kumar Vaibhav

Investigating magnesium-dependent changes in an archaeal RNase P RNA using single-molecule label-free, nanopore sensing and atomic force microscopy

Kumar Vaibhav (Department of Chemistry and Biochemistry, Biophysics Graduate Program, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), William K. Yost (Department of Mechanical and Aerospace Engineering, Biophysics Graduate Program, The Ohio State University, Columbus, OH 43210), Gunjan Agarwal (Department of Mechanical and Aerospace Engineering, Biophysics Graduate Program, The Ohio State University, Columbus, OH 43210), Y. M. Nuwan D. Y. Bandara (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Venkat Gopalan (Department of Chemistry and Biochemistry, Biophysics Graduate Program, Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
RNase P is an essential ribonucleoprotein that employs an RNA catalyst to excise the 5′ leader from precursor (pre) tRNAs to generate mature tRNAs. RNase P RNA (RPR) requires Mg²⁺ for folding and activity. Previous ensemble FRET studies of the RPR from Pyrococcus furiosus (Pfu, an archaeon) uncovered Mg²⁺-dependent inter-domain interactions necessary for pre-tRNA cleavage.1 These ensemble measurements report an average response, thus leaving structural heterogeneity ill-defined. Here, we examined Mg²⁺-dependent changes in Pfu RPR using solid-state nanopores (quartz-based nanopipette sensing) and atomic force microscopy (AFM2) imaging. Nanopores offer a high-throughput real-time single-molecule sensing method that measures transient electrical blockades (i.e., events) caused by RNA translocation through a nanoscale aperture separating two electrolyte reservoir.3 We measured the fraction of nanopore current blocked and the duration of each event from 0 to 500 mM Mg²⁺ and compared their distributions across multiple recordings. We found that the highest [Mg²⁺] tested produced shallower, normalized blockades than the lowest [Mg²⁺]. These findings reveal a reproducible Mg²⁺-dependent change in the nanopore signals and complement observations with ensemble FRET. To assign distinct RNA conformations to the nanopore-sensing events, we undertook AFM analysis which revealed both extended and globular features at low [Mg²⁺] and more compact structures as well as larger clustered features at high [Mg²⁺]. Our ongoing studies using mass photometry are designed to investigate whether RPR oligomerization contributes to the larger clusters. Overall, through use of single-molecule studies to identify a reproducible response of unlabeled RPR to Mg²⁺, this work complements the earlier RPR ensemble FRET-activity relationship analyses,1 and more broadly expands the toolkit available to examine RNA structural re-organization.

References:
1. Marathe IA, Lai SM, Zahurancik WJ, Poirier MG, Wysocki VH, Gopalan V. (2021) Protein cofactors and substrate influence Mg²⁺-dependent structural changes in the catalytic RNA of archaeal RNase P. Nucleic Acids Res 49, 9444–9458.

2. Lee Y-T, Degenhardt MFS, Skeparnias I, Degenhardt HF, Bhandari YR, Yu P, Stagno JR, Fan L, Zhang J, Wang Y-X. (2025) The conformational space of RNase P RNA in solution. Nature 637, 1244–1251.

3. Platnich CM, Alexii RE, Keyser UF. (2026) Solid-state nanopore sensing: the next workhorse of biophysical characterization. Nat Methods, doi: 10.1038/s41592-026-03222-7.

Keywords: Nanopore sensing, RNase P, single molecule