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Characterizing the Conformational Free-Energy Landscape of RNA Stem-Loops Using Single-Molecule Field-Effect

Sukjin S Jang1, Sarah Dubnik1, Jason Hon1

  • 1Department of Chemistry, Columbia University, 3000 Broadway, New York, New York10027, United States.

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Single-molecule field-effect transistors (smFETs) reveal RNA stem-loop folding occurs over a 1-200 ms timescale. This demonstrates a rugged energy landscape for even simple RNA structures, providing new insights into RNA folding dynamics.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Nanotechnology

Background:

  • RNA stem-loops are crucial structural motifs in complex RNA folding.
  • Previous studies reported conflicting timescales for stem-loop (un)folding kinetics.
  • This discrepancy suggests a complex, rugged energy landscape for RNA folding.

Purpose of the Study:

  • To characterize the conformational free-energy landscape of RNA stem-loops.
  • To utilize single-molecule field-effect transistors (smFETs) for direct observation of RNA folding events.
  • To resolve the timescales and conformational transitions during stem-loop (un)folding.

Main Methods:

  • Development and application of single-molecule field-effect transistors (smFETs).
  • Direct monitoring of tens of thousands of individual RNA stem-loop (un)folding events.
  • High-time-resolution (200 μs) observation of conformational changes without mechanical force or fluorescent reporters.

Main Results:

  • Observed RNA stem-loop (un)folding over a 1-200 ms timescale.
  • Identified transitions between ensembles of unfolded and folded conformations.
  • Revealed at least two sub-populations within the folded state, indicating complex folding pathways.

Conclusions:

  • The observed 1-200 ms timescale suggests RNA unfolds through complete trajectories with prolonged exploration of the energy landscape.
  • Findings confirm an extremely rugged free-energy landscape for even simple RNA structural elements.
  • smFETs provide a powerful and unique method for characterizing RNA conformational free-energy and folding dynamics.