Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

57-POS Board 29 METAL ION-DEPENDENT THERMODYNAMICS OF A KISSING LOOP - GAAA TETRALOOP RNA TERTIARY CONTACT Vanessa Schumann 1 ; Paul Lehmann 1 ; Moritz Schaper 1 ; Lucas Schmidt 1 ; Christian Hübner 2 ; Richard Börner 1 ; 1 Mittweida University of Applied Sciences , Laserinstitut Hochschule Mittweida, Mittweida, Germany 2 University of Lübeck, Institute of Physics, Lübeck, Germany RNA molecules frequently rely on tertiary contacts between distinct structural elements to fold into functional conformations, yet detailed thermodynamic characterization of such intramolecular interactions remains limited for many RNA structures. Here, we investigate a tertiary contact essential for ribosome maturation in Saccharomyces cerevisiae, formed between a GAAA tetraloop and a kissing loop, which acts as a tetraloop receptor. Using ensemble FRET measurements, we simultaneously monitored the folding of the receptor and the intramolecular binding of the tetraloop to screen the folded and unfolded states of the construct across a range of conditions, probing the effects of different linkers (poly(A) vs. poly(U)), monovalent and divalent metal ions (K + , Na + , Mg 2+ , Ca 2+ ), and temperature. Temperature-dependent stability was further assessed by UV/Vis melting curve analysis and combined with isothermal urea-induced chemical denaturation at low temperature to resolve complete stability curves for both receptor folding and receptor–tetraloop binding. The construct was fully characterized with respect to metal ion dependence and thermal stability. Monovalent ions (K + , Na + ) stabilized folding of the receptor, whereas divalent ions (Mg 2+ , Ca 2+ ) stabilized binding of the tetraloop to the receptor, with both ions of the same valency being interchangeable. The linker composition did not determine the overall folding behavior but was required to enable intramolecular binding, with measurable differences observed between linker sequences. Together, these results establish an in-depth, high-throughput framework for the thermodynamic characterization of ribosomal RNA systems. Extending this framework with single-molecule FRET will further resolve the underlying folding dynamics, providing complementary insights into the kinetic pathways of RNA folding.

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